Acinetobacter bainite and application thereof

Abstract

The invention provides acinetobacter baylyi and an application thereof, and particularly provides acinetobacter baylyi Ab-ZEN2 which has high screening flux and knockout of hpxO gene and an application of the acinetobacter baylyi Ab-ZEN2, and the preservation number of the acinetobacter baylyi Ab-ZEN2 is GDMCC No: 67727. The strain can be used for testing whether an alternative gene coding region has urate oxidase activity or not, and has higher screening flux.

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 is replaced by SEQ ID NO: 25.

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

[0009] In another aspect, the present disclosure provides an Acinetobacter baumannii preparation comprising the Acinetobacter baumannii Ab-ZEN2 as described above, which is a solid or liquid preparation.

[0010] In another aspect, the present disclosure provides the use of the Acinetobacter Ab-ZEN2 as described above in screening a gene coding region having uricase activity.

[0011] In some embodiments, the uricase comprises a FAD-independent uricase and a FAD-dependent uricase, wherein the FAD-dependent uricase is selected from the group consisting of HpxO type and HpyO type, and the FAD-independent uricase is selected from the group consisting of Pegloticase, Rasburicase and SpUricase.

[0012] In another aspect, the present disclosure provides a method for screening uricase activity, introducing a gene fragment encoding the uricase into the Acinetobacter baumannii Ab-ZEN2 as described above, culturing the bacterial cells in a uric acid medium containing kanamycin, and the growth of the bacterial cells represents the uricase activity.

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

[0014] Advantages The present disclosure provides an Acinetobacter Ab-ZEN2, which has a preservation number of GDMCC No: 67727 and is preserved in the Guangdong Microbial Culture Collection Center. The transformation efficiency of the Ab-ZEN2 strain is about 10 times higher than that of the Ab-HPXOKO1 strain obtained by conventional methods, which can support a higher throughput screening system, and has a higher natural transformation rate to exogenous DNA compared to the same batch of strains. BRIEF DESCRIPTION OF DRAWINGS

[0015] The present disclosure can be more fully understood with reference to the following drawings.

[0016] Figure 1 A flow chart showing the construction 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 An experimental flowchart showing the use of the Ab-ZEN2 strain to screen for genes with uricase activity is shown.

[0032] Figure 17 An experimental flowchart showing the use of the Ab-ZEN2 strain to screen for genes with uricase activity is shown.

[0033] Figure 18 A plate showing transformation of Ab-ZEN2 with Golden Gate Assembly products is shown. DETAILED DESCRIPTION

[0034] The following description of the disclosure is merely intended to illustrate various different embodiments of the disclosure. Thus, the particular modifications discussed should not be construed as limiting the scope of the disclosure. It is apparent to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the disclosure, and it should be understood that such equivalent embodiments are intended to be included within this text. All references cited herein, including publications, patents and patent applications, are incorporated 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 the present application includes naturally occurring protein sequences or variants, truncations thereof. 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 the present application can also be of different origins, for example, derived from bacteria, fungi, mammals. In some embodiments, the Urate Oxidase is derived from human, chimpanzee, orangutan, gorilla, gibbon, baboon, macaque, cynomolgus monkey, night monkey, domestic rabbit, mouse, dog, cow, pig. In some embodiments, the Urate Oxidase is a naturally occurring Urate Oxidase (Uricozyme) derived from Aspergillus flavus. In some embodiments, the Urate Oxidase is a recombinant enzyme, for example, recombinant Aspergillus flavus Urate Oxidase - Rasburicase, Pegloticase. In some specific embodiments, the Urate Oxidase includes FAD-independent urate oxidase (EC 1.7.3.3), FAD-dependent urate oxidase (EC 1.14.13.113), and membrane-integrated uricases. In some embodiments, the FAD-dependent uricases can be of HpxO and HpyO types. The gene (NCBI GeneID: 45235716, https: / / www.ncbi.nlm.nih.gov / gene / 45235716) in Acinetobacter baylyi encodes a FAD-dependent urate oxidase that is capable of converting uric acid to 5-hydroxyisourate (5-HIU), which can spontaneously convert to allantoin under physiological conditions. hpxO

[0036] Acinetobacter baylyi can grow using uric acid as the sole nitrogen source, and the uric acid medium thereof is shown in Table 2, wherein the formulation of 1000X SL-10 solution is shown in Table 1. The ammonium chloride medium of Acinetobacter baylyi using ammonium chloride as the sole nitrogen source is shown in Table 3. The primer sequences used in the specific embodiments of the present application 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] Embodiment In order 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 a part of the embodiments of the present disclosure, not all.

[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 (Ab bacteria) (ATCC number: 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, OG-HpxO-KO-F2 / OG-HpxO-d1261-rev hpxO The left and right homologous arms upstream and downstream of the gene coding region, and the electrophoretogram of the left and right homologous arms are shown in Figure 2 . The left and right homologous arms, 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. Figure 3 Figure 4 hpxO) and Bsal Type IIS restriction enzyme (New England Biolabs, Cat. No. R3733) according to the reaction system of Table 6 and the reaction conditions of Table 7.

[0044] Table 6. Golden Gate Assembly reaction system

[0045] Table 7. Golden Gate Assembly reaction conditions

[0046] a. Wild type Ab strain transformation: Take 70 μL of wild type Ab strain cultured overnight in LB medium, add to a 1 mL fresh LB medium in a shaking tube, and incubate at 30 °C with 200 rpm shaking for 2 hours. Add all 20 μL of the Golden Gate Assembly reaction system to the culture, and continue to incubate at 30 °C with 200 rpm shaking for 2 hours. Collect the bacterial cells, and spread them on a plate containing LB solid medium with 30 μg / mL kanamycin at an appropriate ratio (such as 1:1000), as shown in Figure 5 After the plate is incubated at 30 °C overnight, single colonies are picked and colony PCR is performed to identify whether the expected gene editing has successfully occurred at the genomic location.

[0047] b. Colony PCR identification of correct and successful insertion of DNA fragments: Single colonies are picked from the plate and resuspended with 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, Cat. No. KK5103), and the corresponding electropherogram is shown in Figure 6 The No. 2 bacterial suspension with the correct PCR band is inoculated into LB medium, and after incubation at 30 °C with 200 rpm shaking overnight, the experiment is continued or glycerol is added to a final concentration of 25% and stored at -80 °C.

[0048] 1.2 Ab-HPXOKO1→Ab-HPXOKO2 strain construction (1) pZEN1.1-BB plasmid construction pUC57-Kan plasmid was from GeneWiz. The plasmid backbone fragment of pZEN1.1 (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 Ab strain, and the left and right homologous arms upstream and downstream of the gene coding region were amplified using KAPA HiFi HotStart DNA polymerase and primers OG-pZEN-make-F2-2 / OG-pZEN-make-R2, OG-pZEN-make-F3 / OG-pZEN-make-R3-2. hpxO

[0050] The PCR product electrophoresis is shown in Figure 6 The PCR product was purified using AmPure Xp magnetic beads, and three fragments pZen1.1-Frag1, pZen1.1-Frag2 and pZen1.1-Frag3 were obtained, and Gibson Assembly reaction was performed using NEB Gibson Assembly MasterMix (New England Biolabs, Cat. No. E2611S), and then transformed into DH5a chemically competent cells and plated on LB solid medium containing 30 μg / mL kanamycin. After incubation at 37°C overnight, single colonies were picked and colony PCR was performed to identify whether the expected plasmid pZEN1.1-BB was present.

[0051] A single colony was picked from the plate and resuspended with 10 μL water. 1 μL of the bacterial suspension was taken as a PCR template, and colony PCR was performed using primers pZEN1.1-F1-2 / pZEN1.1-R1-2 and KAPA 2G Fast Genotyping Mix. The colony PCR electrophoresis is shown in Figure 7 As shown in

[0052] (2) Construction of Ab-HPXOKO2 strain ​Ab-HPXOKOl bacteria transformation: 1000 ng of pZEN1.1-BB plasmid was added into 50 μL reaction system, followed by Pmel restriction enzyme (New England Biolabs, Cat# R0560S) and corresponding enzyme buffer, and linearized at 37°C for 2 hours. The DNA fragment with homologous arms at both ends was obtained. hpxO Genomic DNA was extracted from wild type Ab bacteria, and the left and right homologous arms flanking the transposon IS1236 were amplified using KAPA HiFi HotStart DNA polymerase and primers OG-IS1KO-F1 / OG-IS1KO-R1, OG-IS1KO-F2 / OG-IS1KO-R2. The PCR results are shown in Figure 8 70 μL of Ab-HPXOKOl bacteria (No. 2 in Figure 9 ) cultured overnight in LB medium was added into a 1 mL fresh LB medium in a shaking tube, and cultured at 30°C with 200 rpm shaking for 2 hours. The whole 20 μL Pmel enzyme digestion reaction system was added into the culture, and the culture was continued to be incubated at 30°C with 200 rpm shaking for 2 hours. The bacteria were collected and spread onto LB solid medium containing 25 mg / mL Azidothymidine at an appropriate ratio (such as 1:1000), as shown in After overnight incubation at 30°C, single colonies were picked and subjected to colony PCR to identify whether the expected gene editing had successfully occurred at the genomic locus.

[0053] Colony PCR identification Figure 8 Correct gene editing at the locus: single colonies were picked from the plate and resuspended with 10 μL water. 1 μL of the bacterial suspension was taken as the PCR template, and colony PCR was performed using primers Forward Primer 1 / Reverse Primer 1 and KAPA 2G Fast Genotyping Mix. The PCR electrophoretogram is shown in Figure 10 The bacterial suspension of No. 6 with the correct PCR band was inoculated into LB medium, and after overnight incubation at 30°C with 200 rpm shaking, the experiment was continued or glycerol was added to a final concentration of 25% and stored at -80°C.

[0054] 1.3 Ab-HPXOKO2→Ab-ZEN2 strain construction Genomic DNA was extracted from wild type Ab bacteria, and the left and right homologous arms flanking the transposon IS1236 were amplified using KAPA HiFi HotStart DNA polymerase and primers OG-IS1KO-F1 / OG-IS1KO-R1, OG-IS1KO-F2 / OG-IS1KO-R2. The PCR results are shown in Figure 11 Golden Gate Assembly reaction was performed using the left and right homologous arms, pBTK622 plasmid, and Bsal Type IIS restriction enzyme (New England Biolabs, Cat# R3733) 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 strain transformation: Take 70 μL of Ab-HPXOKO2 strain (No. 6 in Table 7) cultured overnight in LB medium, add to a 1 mL fresh LB medium in a shaking tube, and incubate at 30 °C with 200 rpm shaking for 2 hours. Add the whole 20 μL of Golden Gate Assembly reaction system to the culture, and continue incubation at 30 °C with 200 rpm shaking for 2 hours. Collect the bacterial cells, spread on LB solid medium containing 30 μg / mL kanamycin at an appropriate ratio (such as 1:1000), and incubate at 30 °C overnight. After incubation, pick single colonies and perform colony PCR to identify whether the desired gene editing has successfully occurred at the genomic locus. hpxO Figure 12

[0057] Colony PCR to identify the correct and successful insertion of DNA fragment: Pick a single colony from the plate and resuspend with 10 μL water. Take 1 μL of the bacterial suspension as the PCR template, and perform colony PCR using primers Forward Primer 2 / Reverse Primer 2 and KAPA 2G Fast Genotyping Mix. The corresponding electropherogram is shown in Figure 13 . Inoculate the bacterial suspension with the correct PCR band into LB medium, incubate at 30 °C with 200 rpm shaking overnight, and continue the experiment or add glycerol to a final concentration of 25% and store at -80 °C. Extract the genomic DNA from the overnight cultured bacterial cells, and use Sanger sequencing to determine whether the DNA sequence at the desired position is as expected. Colony No. 1 is finally selected as the Ab-ZEN2 strain.

[0058] Sanger sequencing peak chart showing that the coding region of the hpxO gene in the Ab-ZEN2 strain has been successfully deleted and replaced by a 26 base pair sequence ref: Suárez , and the kanamycin resistance gene expression cassette has replaced the original transposon IS1236 gene sequence at the transposon IS1236 locus in the expected manner 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. .

[0059] In summary, in the Ab-ZEN2 strain, Figure 1 ​​The 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 ( Figure 11 hpxO 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. Figure 11 Figure 11 Its construction process is as follows: Figure 14 As shown.

[0060] Example 2: Phenotypic Analysis of Ab-ZEN2 Strains 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...) hpxO 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 successfully transformed cells with complementary phenotypes. 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. ​ Methods for constructing Ab bacteria with genes ( ​ ​ ​ ​) is equivalent. From the experimental data, it can be seen that the transformation efficiency of the selected Ab-ZEN2 strain clone (No. 1 colony in ​ is as high as 2.93 x 10 8 cfu per µg DNA, which is significantly higher than 1.27 x 10 7 cfu per µg DNA of the Ab-HPXOKO1 strain used for comparison, more than 10 times higher, and also significantly higher than some other clones obtained in the same batch, such as No. 2-7 colonies (the transformation efficiency is equivalent to the Ab-HPXOKO1 strain, 1.45 x 10 7 cfu per µg DNA) (Table 9). This makes it possible to have higher screening throughput when using the selected Ab-ZEN2 strain as a chassis cell to screen a DNA library containing a large number of different coding regions, or to reduce the experimental scale and cost under the same screening throughput.

[0063] Therefore, when using a DNA with a 1000 base pair left homologous arm and right homologous arm for transformation, the Ab-ZEN2 strain retains the natural transformation ability of the wild type Ab strain. Unexpectedly, different Ab-ZEN2 strains have different transformation efficiencies, and the transformation efficiency of the finally selected Ab-ZEN2 strain is about 10 times higher than that of the Ab-HPXOKO1 strain obtained by conventional methods, so it can support a higher throughput screening system.

[0064] Different Ab-ZEN2 strain clones obtained in the same batch have different natural transformation rates. The finally selected Ab-ZEN2 clone has unexpectedly high natural transformation rate to exogenous DNA, which is relatively high among the Ab-ZEN2 clones obtained in the same batch, and has a higher natural transformation rate to exogenous DNA than the Ab-HPXOKO1 strain obtained by conventional methods.

[0065] Table 9. Transformation ratio per viable cell number and transformation efficiency per exogenous DNA dosage

[0066] 2.2 Bacterial strain preservation The Ab-ZEN2 strain (colony 1 of ​ has been preserved at the Guangdong Microbial Culture Collection Center (GDMCC), located at No. 59, Building 5, 100, Martyrs' Road, Guangzhou, which is a national patent strain preservation institution and an international preservation unit under the Budapest Treaty. The preservation number of the Ab-ZEN2 strain is GDMCC No: 67727, the preservation time is January 22, 2026, and the classification name is Acinetobacter baylyi.

[0067] 2.3 Ab-ZEN2 stock 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 complemented strain: Ab-ZEN2-cmp strain construction The construction process of Ab-ZEN2 complemented strain Ab-ZEN2-cmp is shown in ​ and the specific operation is as follows: Genomic DNA was extracted from wild type Ab strain, and KAPA HiFi HotStart DNA polymerase and primers OG-HpxO-u1177-fwd / OG-HpxO-d1261-rev were used to amplify the DNA fragment with homologous arms and gene coding region sequence. ​

[0068] Take 70 μL of Ab-ZEN2 stock strain cultured overnight in LB medium and add it to a 1 mL fresh LB medium in a shaking tube, and cultivate at 30°C, 200 rpm for 2 hours. Add 100 ng of PCR product to the culture, and continue to cultivate at 30°C, 200 rpm for 2 hours. Collect the bacterial cells, and plate them on a plate containing 30 μg / mL kanamycin in uric acid solid medium at an appropriate ratio (such as 1:1000). After the plate is incubated at 30°C for 48-96 hours, single colonies are picked and used as Ab-ZEN2 complemented strain Ab-ZEN2-cmp for further experiments.

[0069] (2) Growth curve determination of Ab-ZEN2 and Ab-ZEN2-cmp Three single colonies of Ab-ZEN2 and Ab-ZEN2-cmp were picked respectively, and first cultivated overnight at 30°C, 300 rpm in ammonium chloride medium containing 30 μg / mL kanamycin to the platform phase. The next morning, 500 μL of each of the 6 cultures were washed twice with 1×PBS to prevent the entry of ammonium chloride into the subsequent uric acid medium, and then resuspended in 500 μL of 1×PBS. 2 μL of the resuspension was inoculated into 3 mL of ammonium chloride medium containing 30 μg / mL kanamycin or 3 mL of uric acid medium containing 30 μg / mL kanamycin in a 10 mL shaking tube, and cultivated at 30°C, 300 rpm. The OD 600 of each culture was measured at 0 hours, 12 hours, 14 hours, 16 hours, 18 hours, 23 hours, 36 hours, 44 hours and 60 hours to determine the growth.

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

[0071] The results are shown in Table 10 and Figure 15 Ab-ZEN2 strain can grow in ammonium chloride medium containing 30 pg / mL kanamycin, but cannot grow in uric acid medium containing 30 pg / mL kanamycin. After transforming the coding region DNA of wild-type hpxO gene having uric acid oxidase activity into Ab-ZEN2 strain, the phenotype that it cannot grow in uric acid medium can be complemented. After complementation, Ab-ZEN2-cmp strain can grow in both ammonium chloride medium containing 30 pg / mL kanamycin and uric acid medium containing 30 pg / mL kanamycin.

[0072] 2.4 Screening of gene coding region having uric acid oxidase activity using Ab-ZEN2 strain FAD-dependent uric acid oxidase, such as HpxO type uric acid oxidase, and FAD-independent uric acid oxidase (such as Pegloticase, Rasburicase, and SpUricase of Schizosaccharomyces pombe) have different reaction mechanisms with uric acid. FAD-independent uric acid oxidase uses oxygen molecules to directly oxidize uric acid, while producing 5-HIU and hydrogen peroxide (H2O2), while HpxO type uric acid oxidase uses NADH or NADPH as a co-substrate in addition to oxygen molecules, producing NAD + and NADP + in addition to 5-HIU, but does not produce hydrogen peroxide (H2O2).

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

[0074] The CDS of SpUricase gene from Schizosaccharomyces pombe was added with left and right homology arms in a screening manner, and the DNA was used to transform Ab-ZEN2 stock strain, which can complement hpxOPhenotype of gene knockout, the Ab-ZEN2 strain after transformation can grow in uric acid medium.

[0075] An example of experiment using Ab-ZEN2 chassis cell to test whether an alternative gene coding region has uric acid oxidase activity is as follows (operation flow as shown in Figure 16 and Figure 17 ): Extract genomic DNA of Schizosaccharomyces pombe, use KAPA HiFi HotStart DNA polymerase and primers SpUO-U0-2 / SpUO-D888-2 to amplify DNA fragment of uric acid oxidase gene SpUricase in Schizosaccharomyces pombe (SEQ ID NO: 34). Perform Golden Gate Assembly reaction of DNA fragment of SpUricase and pZEN1.1-BB plasmid according to reaction system as shown in Table 11 and reaction conditions of Table 7 to add homologous arms of hpxO locus to upstream and downstream of SpUricase coding region.

[0076] Table 11. Reaction system of Golden Gate Assembly

[0077] Use Golden Gate Assembly product to transform Ab-ZEN2: take 70 μL of Ab-ZEN2 bacteria cultured overnight in LB medium, add to 1 mL of fresh LB medium in a shaking tube, cultivate at 30°C with 200 rpm shaking for 2 hours. Add all 25 μL of Golden Gate Assembly product to the culture, continue to cultivate at 30°C with 200 rpm shaking for 2 hours. Collect the bacteria, take 1 / 1000 dilution with sterile water and spread on a plate containing 30 μg / mL of kanamycin in uric acid solid medium, as shown in Figure 18 . Observe the growth of colonies after 48-72 hours of cultivation at 30°C. The growth of colonies indicates that the SpUricase gene from Schizosaccharomyces pombe has uric acid oxidase activity.

[0078] The results are shown in Figure 18 : The results prove that the SpUricase gene from Schizosaccharomyces pombe (S. Pomb) can complement the hpxO -KO phenotype in Ab-ZEN2 strain, proving that Ab-ZEN2 strain can be used to screen gene coding regions with uric acid oxidase activity.

[0079] By reference The entire contents of each patent and scientific document referred to herein is incorporated by reference for all purposes.

[0080] Equivalents The present disclosure can be embodied in other specific ways without departing from the spirit or essential characteristics thereof. Therefore, the above embodiments are to be considered in all respects as illustrative and not restrictive, and the scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. Acinetobacter baylyi Ab-ZEN2, with the preservation number of GDMCC No: 67727.

2. The Acinetobacter baylyi Ab-ZEN2 of claim 1, comprising a deletion of the urate oxidase gene. hpxO of claim 1, comprising a deletion of the urate oxidase gene.

3. The Acinetobacter baylyi Ab-ZEN2 of claim 1 or 2, comprising a kanamycin resistance gene.

4. The Acinetobacter baylyi Ab-ZEN2 of claim 1, comprising a nucleic acid sequence as shown in SEQ ID NO:

25.

5. The Acinetobacter baylyi Ab-ZEN2 of claim 1, comprising a nucleic acid sequence as shown in SEQ ID NO:

26.

6. An Acinetobacter baylyi preparation, comprising the Acinetobacter baylyi Ab-ZEN2 of any one of claims 1 to 5, which is a solid or liquid preparation.

7. Use of the Acinetobacter baylyi Ab-ZEN2 of any one of claims 1 to 5 in screening a gene coding region with uric acid oxidase activity.

8. The use of claim 7, wherein the uric acid oxidase comprises a FAD-independent uric acid oxidase and a FAD-dependent uric acid oxidase.

9. A method for screening uric acid oxidase activity, introducing a gene fragment encoding the uric acid oxidase into the Acinetobacter baylyi Ab-ZEN2 of any one of claims 1 to 5, culturing the bacterial cells in a uric acid medium containing kanamycin, and the growth of the bacterial cells represents the uric acid oxidase activity.

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

Images