Patent Application
20250163479
This application claims priority to Chinese patent application No. 2022113183630, titled “VISCOUS POLYHYDROXYALKANOATE, PREPARATION THEREOF, AND USE THEREOF”, filed on Oct. 26, 2022. The contents of the above identified applications are hereby incorporated herein in their entireties by reference.
The present application relates to the field of microbiological technology, and particularly relates to a viscous polyhydroxyalkanoate, and its preparation and use.
Polyhydroxyalkanoates (PHAs) are polyesters intracellularly synthesized in a variety of microorganisms, and primarily found as storage materials for carbon sources and energy in organisms. PHAs possess physical and chemical properties similar to synthetic plastics, as well as superior features not found in synthetic plastics such as biodegradability, biocompatibility, optical activity, piezoelectricity, and gas barrier property. PHAs have broad application prospects in biodegradable packaging materials, tissue engineering materials, controlled-release materials, electrical materials, and medical materials.
There are methods for preparing conventional PHAs, such as those disclosed in CN114480317A, CN114480318A, etc. However, the varieties of conventional PHAs are limited, and viscous PHAs have not been reported.
The present application is filed in view of the above.
Based on this, according to various embodiments of the present application, a viscous PHA and its preparation and use are provided. The technical solutions are as follows:
In a first aspect of the present application, a mutant of PHA polymerase PhaC61-3 is provided. The mutant has following amino acid mutation sites: E115T, A261E, R360S, and K457Q. The PHA polymerase PhaC61-3 has an amino acid sequence as set forth in SEQ ID No. 9.
In a second aspect of the present application, a nucleic acid fragment is provided. The nucleic acid encodes the mutant.
In a third aspect of the present application, a recombinant vector is provided. The vector includes the nucleic acid fragment.
In a fourth aspect of the present application, a genetically engineered bacterium is provided. The bacterium is Escherichia coli containing the recombinant vector, or the bacterium is Pseudomonas having a genome into which the nucleic acid fragment is integrated.
In some embodiments of the present application, the Escherichia coli is strain S17-1.
In some embodiments of the present application, the Pseudomonas is strain PE1668.
In a fifth aspect of the present application, a viscous PHA is provided, which has the following structural formula:
In a sixth aspect of the present application, use of the viscous PHA in preparation of a degradable product is provided.
In a seventh aspect of the present application, a degradable product is provided, which includes the viscous PHA.
In an eighth aspect of the present application, use of the mutant, the nucleic acid fragment, the recombinant vector, or the Escherichia coli in preparation of the viscous PHA is provided.
In a ninth aspect of the present application, a method for preparing the viscous PHA is provided. The preparation method includes following steps:
In some embodiments of the present application, the fermentation culture medium includes 0 g/L to 40 g/L glucose, 0.1 g/L to 5 g/L 10-undecenoic acid, and lysogeny broth (LB) medium.
In some embodiments of the present application, the fermentation culture medium includes 10 g/L to 40 g/L glucose, 1 g/L to 3.5 g/L 10-undecenoic acid, and LB medium.
In some embodiments of the present application, the fermentation culture is performed at a temperature of 28° C. to 42° C. for a time period of 12 hours (h) to 72 h with a rotation speed of 150 rpm to 300 rpm.
Details of one or more embodiments of the present application are set forth in the following description, and other features, objectives, and advantages of the present application will become apparent from the specification and claims.
In order to illustrate the embodiments of the present disclosure more clearly, the drawings used in the embodiments will be described briefly. Apparently, the following described drawings are merely for the embodiments of the present disclosure, and other drawings can be derived by those of ordinary skill in the art without any creative effort.
The technical solutions in embodiments of the present application will now be clearly and fully described in detail with reference to the accompanying drawings. The embodiments described herein are only some but not all embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the scope of protection of the present application.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application pertains. The terms used in the specification of the present application are merely for the purpose of describing specific embodiments and are not intended to limit the present application.
The present application provides a mutant of PHA polymerase PhaC61-3, and the mutant has following amino acid mutation sites: E115T, A261E, R360S, and K457Q. The amino acid sequence of PHA polymerase PhaC61-3 is as shown in SEQ ID No. 9.
The present application provides a nucleic acid fragment, and the nucleic acid encodes the mutant.
The present application provides a recombinant vector including the nucleic acid fragment.
The present application provides a genetically engineered bacterium. The bacterium is Escherichia coli containing the recombinant vector, or the bacterium is Pseudomonas having a genome into which the nucleic acid fragment is integrated.
In some embodiments of the present application, the Escherichia coli is strain S17-1.
In some embodiments of the present application, the Pseudomonas is strain PE1668.
The present application provides a viscous PHA, which has the following structural formula:
The present application provides use of the viscous PHA in preparation of a degradable product.
The present application provides a degradable product including the viscous PHA.
The degradable product, for example, is a packaging material, a tissue engineering material, a controlled-release material, an electrical material, or a medical material.
The present application provides use of the mutant, the nucleic acid fragment, the recombinant vector, or the Escherichia coli in preparation of the viscous PHA.
The present application provides a method for preparing the viscous PHA, which includes the following steps:
In some embodiments, the fermentation culture medium includes 0 g/L to 40 g/L glucose, 0.1 g/L to 5 g/L 10-undecenoic acid, and LB medium. The fermentation culture medium can include glucose or be free of glucose. Glucose can have a concentration (in g/L), such as 0 g/L, 2 g/L, 4 g/L, 6 g/L, 8 g/L, 10 g/L, 12 g/L, 14 g/L, 16 g/L, 18 g/L, 20 g/L, 22 g/L, 24 g/L, 26 g/L, 28 g/L, 30 g/L, 32 g/L, 34 g/L, 36 g/L, 38 g/L, or 40 g/L. 10-undecenoic acid can have a concentration (in g/L) such as 0.1 g/L, 0.5 g/L, 1 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, or 5 g/L.
In some embodiments, the fermentation culture medium includes 10 g/L to 40 g/L glucose, 1 g/L to 3.5 g/L 10-undecenoic acid, and LB medium.
In some embodiments, the fermentation culture is performed at a temperature of 28° C. to 42° C. for a time period of 12 hours (h) to 72 h with a rotation speed of 150 rpm to 300 rpm. The fermentation temperature (in ° C.) can be such as 28° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., or 42° C. The fermentation time period (in hour) can be such as 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, 56 h, 60 h, 64 h, 68 h, or 72 h. The rotation speed (in rpm) can be such as 150 rpm, 175 rpm, 200 rpm, 225 rpm, 250 rpm, 275 rpm, or 300 rpm.
The following examples are provided to specifically describe the embodiments of the present application. It should be understood that the examples are for illustration only and not intended to limit the scope of the present application. In the following examples, experimental methods not specifying specific conditions should refer first to the guidance given in the present application, or to the experimental manuals or conventional conditions in the art, or to the conditions recommended by manufacturers, or to the experimental methods known in the art.
In the specific examples described below, the measurement parameters for raw material components may have minor deviations within the range of weighing accuracy unless specifically stated, and the temperature and time parameters may have acceptable deviations due to instrument testing accuracy or operational precision.
(1) Comparison of PHA Polymerase Amino Acid Sequences from Different Pseudomonas Strains
The protein sequence of PHA polymerase (PhaC61-3, from Pseudomonas sp.61-3) in recombinant Pseudomonas PE1668 (Li M, Ma Y, Zhang X, Zhang L, Chen X, Ye J W, Chen G Q. Tailor-Made Polyhydroxyalkanoates by Reconstructing Pseudomonas Entomophila. Adv Mater. 2021 October; 33 (41): e2102766. doi: 10.1002/adma.202102766. Epub 2021 Jul. 28. PMID: 34322928) was analyzed using NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to screen out strains with high homology, similar structures, and identical functions: Pseudomonas sp. Irchel 3E20, Pseudomonas sp. H3, Pseudomonas sp. GM17, Pseudomonas sp. SK3, and Pseudomonas sp. SWRI18. The amino acid sequences of these strains and PhaC61-3 were compared using MEGA 6.0 software and the website https://espript.ibcp.fr. The specific comparison results are shown in
The PHA polymerase PhaC61-3 (of strain PE1668) has amino acid differences only at positions 325 and 481 as compared to the native sequence of PhaC61-3 (from Pseudomonas sp. 61-3), but includes more differences as compared to the other strains. For these differences, the following four amino acid sites were mutated: E115T, A261E, R360S, K457Q. Primers were designed as follows for these mutations, and the required gene fragments were obtained and connected using T4 ligase.
{circle around (1)} For the mutation E115T, primers were as follows, designed using Snapgene software:
The reaction system was in a total volume of 50 μL, and includes the following components added in a 0.2 mL PCR tube:
After mixing and instant centrifugation, the reaction parameters are: denaturation at 98° C. for 30 seconds (sec); 35 cycles for denaturation at 98° C. for 10 sec, annealing at 72° C. for 30 sec, and extension at 72° C. for 5.5 minutes (min); and a final extension at 72° C. for 2 min.
The formed gene fragments were collected using a universal DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.), following the steps provided in the product manual.
The gene fragments were ligated using T4 DNA Ligase (purchased from New England Biolabs) in a reaction system with a total volume of 10 μL, including the following components sequentially added in a 0.2 mL PCR tube:
After mixing and instant centrifugation, the ligation was carried out overnight at 16° C. to obtain Ligated Product 1.
{circle around (2)} For the mutation A261E, the following operations were performed on Ligated Product 1 obtained in step {circle around (1)}:
The primer sequences designed using Snapgene software and Q5 system reaction parameters were as follows:
Reaction parameters: denaturation at 98° C. for 30 sec; 35 cycles for denaturation at 98° C. for 10 sec, annealing at 72° C. for 30 sec, and extension at 72° C. for 5.5 min; and a final extension at 72° C. for 2 min.
The obtained gene fragments were ligated using the T4 ligase in the same method as in step {circle around (1)}, resulting in Ligated Product 2.
{circle around (3)} For the mutation R360S, the following operations were performed on Ligated Product 2 obtained in step {circle around (2)}:
The primer sequences designed using Snapgene software and Q5 system reaction parameters were as follows:
Reaction parameters: denaturation at 98° C. for 30 sec; 35 cycles for denaturation at 98° C. for 10 sec, annealing at 72° C. for 30 sec, and extension at 72° C. for 5.5 min, and a final extension at 72° C. for 2 min.
The obtained gene fragments were ligated using the T4 ligase in the same method as in step {circle around (1)}, resulting in Ligated Product 3.
{circle around (4)} For the mutation K457Q, the following operations were performed on Ligated Product 3 obtained in step {circle around (3)}:
The primer sequences designed using Snapgene software and Q5 system reaction parameters were as follows:
Reaction parameters: denaturation at 98° C. for 30 sec; 35 cycles for denaturation at 98° C. for 10 sec, annealing at 72° C. for 30 sec, and extension at 72° C. for 5.5 min; and a final extension at 72° C. for 2 min.
The obtained gene fragments were ligated using the T4 ligase in the same method as in step {circle around (1)}, to finally obtain Ligated Product 4.
(3) Preparation of Escherichia coli S17-1 Competent Cells
{circle around (1)} A LB culture plate was used, and Escherichia coli S17-1 (preserved at −80° C. in glycerol) was picked with an inoculating loop and streaked into the plate, which was incubated at 37° C. for 20 h to 24 h.
{circle around (2)} A single colony of Escherichia coli S17-1 was picked from the LB plate and inoculated into a shake tube (5 mL of LB liquid medium) and incubated with shaking at 37° C. for 12 h.
{circle around (3)} The above culture was inoculated into a 150 ml conical flask (LB liquid medium) at 1% (v/v), incubated with shaking at 37° C. until OD600 reaches 0.4 to 0.5, then placed on ice to halt the growth.
{circle around (4)} 1 mL of the above culture was transferred into a 1.5 mL centrifuge tube, centrifuged at 4000 rpm, 4° C. for 10 min, then the supernatant was discarded; then operations followed the instructions of the E. coli Competent Cell Preparation Kit (by Takara Company).
{circle around (5)} The competent cells were divided into 50 μL/tube on ice, stored at −80° C., thus obtaining Escherichia coli S17-1 competent cells.
(4) Transformation of Ligated Product into Escherichia coli S17-1
{circle around (1)} 50 μL of Escherichia coli S17-1 competent cells was taken and thaw on ice, added with 5 μL of the final Ligated Product 4 including all four amino acid mutations from step (2), gently mixed, and left on ice for 30 min.
{circle around (2)} The mixture was heat shocked at 42° C. for 60 sec in a water bath, then the tube was quickly transferred to an ice bath for 2 min stay.
{circle around (3)} 500 μL of sterile LB liquid medium (without antibiotics) was added to the mixture, mixed well and incubated at 37° C., 220 rpm for 1 h.
{circle around (4)} 200 μL of the culture from step 3 was spread onto an LB plate containing kanamycin, and incubated at 37° C. for 16 h.
{circle around (5)} A positive single colony was picked and inoculated into a shake tube containing LB liquid medium (5 mL, supplemented with 1% % kanamycin), incubated overnight at 37° C., 220 rpm. The positive single colony was verified by PCR of the culture and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis. Sequence alignment confirmed the successful construction of the recombinant plasmid.
(5) Conjugation Between Escherichia coli S17-1 and Pseudomonas
{circle around (1)} Escherichia coli S17-1 and Pseudomonas entomophila LAC32 cultures, 50 μL for each, were dropped onto a LB plate, and incubated at 30° C. for 10 h.
{circle around (2)} The cultures on the plate were streaked with a pipette tip in a grid pattern (three lines horizontally and three lines vertically), then spread onto an LB plate containing kanamycin (Kan) and chloramphenicol (Cm) using the same pipette tip; then the present plate was added with 200 μL of LB liquid medium, which was spread using a spreading rod, and the plate was incubated at 30° C. for 48 h.
{circle around (3)} The positive monoclonal colonies grown in the plate were streaked onto another LB plate containing Kan and incubated at 30° C. for 24 hours.
{circle around (4)}A single colony was picked and inoculated into a shake tube containing 5 mL LB liquid medium with Kan, incubated at 30° C., 220 rpm for 12 h. The recombinant Pseudomonas, named PE1668-1, was stored at −80° C. in 50% glycerol for future use.
The fermentation production of this novel material was carried out using the recombinant Pseudomonas PE1668-1 containing the plasmid, obtaining wet bacterial biomass containing PHA (P (3HB-co-3HU10D))
{circle around (1)} Monoclonal Colonies: the above recombinant strain PE1668-1 was streaked using an inoculating loop onto an LB plate containing Kan on a clean bench. The plate was incubated at 30° C. for 24 h to allow for the growth of monoclonal colonies.
{circle around (2)} Primary Seed Culture: a monoclonal colony from step {circle around (1)} was picked and inoculated into a shake tube containing 5 mL of seed culture medium (LB+1% % Kan), and cultured at 30° C., 200 rpm for 12 h.
{circle around (3)} Secondary Seed Culture: 200 μL (1% inoculum, v/v) of the culture from step {circle around (2)} was inoculated into a 150 ml conical flask containing 20 mL of seed culture medium (LB+1% % Kan), and incubated at 30° C., 200 rpm for 12 h.
LB Medium: yeast powder 5 g/L; tryptone 10 g/L; sodium chloride 10 g/L;
The seed culture was inoculated at 5% (v/v) into a 2 L shake flask containing 400 mL of the LB medium, and 1% % Kan antibiotic, various concentrations of carbon source, and the seed culture were sequentially added. The culture system was incubated at 30° C., 200 rpm for 48 h.
Cell Dry Weight (CDW): 25 mL of the post-fermentation culture was transferred into a 50 mL centrifuge tube, and centrifuged at 8000 rpm at room temperature for 6 min. The supernatant was discarded, and the precipitate was washed with deionized water twice, frozen in a refrigerator at −80° C. for 1 h, lyophilized in a vacuum freeze-dryer for 18 h, and weighed. The CDW (g/L) was calculated by dividing the resulted weight by the original volume of the culture.
PHA (P (3HB-co-3HU10D)) Content Measurement: 40 mg of the weighed lyophilized cells was added with 2 mL of esterification liquid (containing methanol, 3% (v/v) concentrated sulfuric acid (98%, w/w), and 1 g/L benzoic acid) and 2 mL chloroform, and esterified at 100° C. for about 4 h. 25 mg of a standard PHA with the same treatments was used for reference. Subsequently, the PHA content was measured using a GC-2014 gas chromatograph (Shimadzu, Japan). The test method was as follows: initial temperature was 80° C. and maintained for 1.5 min; in the first phase, the temperature was increased to 140° C. at a rate of 30° C./min; in the second phase, the temperature was increased to 240° C. at a rate of 40° C./min, which took 2 min; the total analysis time was 8 minutes; the injection temperature was 240° C. and the detector temperature was 250° C.
The fermentation results and the content of PHA (P (3HB-co-3HU10D)) vary with different concentrations of 10-undecenoic acid and glucose, as shown in Table 3 (all data in the table are presented as mean±variance):
{circle around (1)} Obtaining Wet Bacterial Biomass:
The Pseudomonas culture after the fermentation in Example 2, step (3) was collected and poured into a 50 mL centrifuge tube, and centrifuged at room temperature for 6 min at 8000 rpm. The supernatant was discarded carefully so as not to lose the precipitate, which was then added with deionized water to resuspend, and centrifuged at room temperature for 6 min at 8000 rpm, and this process was repeated twice.
Cell disruption was achieved using the aqueous phase separation method, wherein a beaker containing the treated bacterial suspension was heated, and once the temperature reached 50° C., 1 mg/mL surfactant was added and the pH was maintained at about 9 with 5M NaOH for 15 min, during which the suspension was stirred continuously.
After cooling to room temperature, the mixture was transferred into multiple 50 mL centrifuge tubes and centrifuged at room temperature for 6 min at 8000 rpm. The supernatant was discarded while minimizing loss of precipitate. 30 mL deionized water was added to resuspend the precipitate while ensuring complete dissolution of the precipitate, The resuspension was centrifuged at room temperature for 6 min at 8000 rpm, and the supernatant was discarded; and this process was repeated twice. The crude PHA (P (3HB-co-3HU10D)) product was frozen at −80° C. for 0.5 h, and lyophilized in a vacuum freeze-dryer for 10 h, and the final product was then weighed.
The extraction and purification of the obtained PHA material were performed according to the method described in the reference Follonier, S., Riesen, R., & Zinn, M. (2015), Pilot-scale Production of Functionalized mcl-PHA from Grape Pomace Supplemented with Fatty Acids, Chemical and Biochemical Engineering Quarterly, 29, 113-121. The method for extraction and purification involved initially using dichloromethane for extraction on the crude PHA product, followed by precipitation and purification with cold methanol. Specifically, the weighed crude PHA product was added with dichloromethane at a ratio of 1 g/17 ml, stirred continuously, and then allowed to settle for 3 h. The mixture was suction filtered to remove the residue, retaining the filtrate containing the PHA material in a suitable beaker. While stirring continuously, cold methanol was added dropwise to methanol volume/dichloromethane volume containing PHA being 5:1. In this process precipitation of the material can be observed. The solution was left to settle for 1 h, filtered to retain the PHA material, which was then redissolved in dichloromethane and dried in a vacuum dryer for 24 h to obtain the final PHA material.
{circle around (1)} Measurement of PHA (P (3HB-co-3HU10D)) Content: The content was determined using gas chromatography, similar to Example 2.
{circle around (2)} Measurement of PHA (P (3HB-co-3HU10D)) Quantity: Considering the viscosity of the material, during purification, the material was disposed in a glass dish or a beaker for evaporation. The mass of the material can be determined by the change in mass Δm, where Δm=mtotal−m0, mtotal is the total mass of the material and the glass dish or beaker, and m0 is the mass of the glass dish or beaker.
The quantity of the purified PHA product is shown in Table 4. The Test No. correspond to the fermentation results in Table 3 (For example, 2-1 in Table 4 corresponds to 1-1 in Table 3), and the data in the table are presented as mean±variance.
The PHA (P (3HB-co-3HU10D)) material extracted and purified in Example 3 was subjected to NMR analysis to determine the proportion of each monomer. As the ratio of the PHA (P (3HB-co-3HU10D)) material varies with different raw material ratios:
When the glucose content was 30 g/L to 34 g/L, and the 10-undecenoic acid content was 1 g/L to 2 g/L, the 3HU10D monomer can reach 15% to 25%. The 1HNMR result is shown in
When the glucose content was 20 g/L to 25 g/L, and the 10-undecenoic acid content was 2 g/L to 2.5 g/L, the 3HU10D monomer can reach 30% to 40%. The 1HNMR result is shown in
When the glucose content was 10 g/L to 17 g/L, and the 10-undecenoic acid content was 2.5 g/L to 3.2 g/L, the 3HU10D monomer can reach 45% to 50%. The 1HNMR result is shown in
In Example 3, the purified material P (3HB-co-3HU10D) exhibited transparent and viscous characteristics, as shown in
Viscosity Testing: 20 mg of the material was applied evenly on one side of a glass sheet A, and pressed with another clean glass sheet B, ensuring that both glass sheets and the material therebetween were in close contact; then a 0.1 g weight was hung on the other side of the glass sheet B using a clip and a rubber band; after assembly, the two glass sheets were vertically arranged and the weight was continuously changed until the glass sheets A and B start to separate. Referring to
The viscosity test on 20 mg of P (3HB-co-3HU10D) materials with different 3HU10D monomer contents was performed, and the maximum weights the material can withstand are as follows (Table 5):
The above data indicate that the higher the proportion of the 3HU10D monomer in the PHA, the greater the viscosity of the material, and the greater the maximum weight the material can withstand. When the proportion of the 3HU10D monomer is above 45%, the maximum weight the material can withstand is 1.67 kg.
{circle around (1)} Underwater Viscosity Analysis with Different Carriers:
The PHA materials with different 3HU10D proportions obtained in Example 3 were analyzed for underwater viscosity.
Different adhered carriers were used for underwater viscosity testing: 20 mg of the material was each applied evenly on glass sheets, metal sheets, plastic sheets, collagen films, and PVA films of the same size, which were then disposed respectively in beakers containing deionized water (at room temperature) for a duration of 0.5 h, followed by the viscosity testing method described in (2).
Underwater viscosity tests were carried out on 20 mg of P (3HB-co-3HU10D) material with different 3HU10D monomer contents, with the maximum weight tolerance as follows (Table 6):
The above data indicate that for the same carrier, the lower the proportion of the 3HU10D monomer, the less the maximum weight the material can withstand; for the same proportion of the 3HU10D monomer, the collagen film as a carrier can withstand the greatest weight, the PVA film is the second, and the glass sheet also shows significant viscosity.
{circle around (2)} Underwater Viscosity Testing at Different Temperatures:
20 mg of the material was each evenly applied on glass sheets, collagen films, and PVA films of the same size, which were then disposed respectively in beakers containing deionized water at different temperatures for a duration of 0.5 h, followed by the viscosity testing method described in (2).
The maximum weight tolerance at different conditions is as follows (Table 7):
The above data indicate that for the same carrier and at the same temperature, the higher the proportion of the 3HU10D monomer, the greater the maximum weight the material can withstand; for the same proportion of the 3HU10D monomer, the collagen film as a carrier at a temperature of 37° C. can withstand the greatest weight, and the PVA film is the second.
The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features are described in the embodiments. However, as long as there is no contradiction in the combination of these technical features, the combinations should be considered as in the scope of the present disclosure.
The above-described embodiments are only several implementations of the present disclosure, and the descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present disclosure. It should be understood by those of ordinary skill in the art that various modifications and improvements can be made without departing from the concept of the present disclosure, and all fall within the protection scope of the present disclosure. Therefore, the patent protection of the present disclosure shall be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202211318363.0 | Oct 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/134612 | 11/28/2023 | WO |
