Pseudoalteromonas winterhai M044 for producing alteromide A and application of pseudoalteromonas winterhai M044

Abstract

The invention relates to the field of biotechnology and microbiology, in particular to Pseudoalteromonas wwarhai M044 for producing alteromide A and application of the Pseudoalteromonas wwarhai M044, and the provided strain can be used for stably synthesizing secondary metabolites with biological activity, such as alteromide A and the like. Functional verification shows that the strain and a purified product thereof have remarkable inhibitory activity on staphylococcus aureus and bacillus subtilis, and the minimum inhibitory concentration of the strain and the purified product thereof is measured; meanwhile, a biosynthetic gene cluster of the strain is successfully positioned through genome analysis, and it is indicated that the strain has a definite synthetic route and stable product synthesis capacity. Compared with a traditional antibacterial compound, the strain and the product of the strain are natural in source, safe, environment-friendly and clear in biosynthesis route, an efficient and controllable production process can be realized, and a new microbial resource and a technical route are provided for developing a novel marine-derived antibacterial agent.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and microbiology, specifically to a strain of Alteramide A-producing Pseudomonas wenyusense M044 and its applications. Background Technology

[0002] With the widespread use and even abuse of traditional antibiotics, antibiotic resistance has become an increasingly serious problem, while the potential toxicity of some synthetic antimicrobial agents limits their application in food, medicine, and other fields. Therefore, developing safe and novel natural antimicrobial products has become an important research direction. Against this backdrop, marine ecosystems, especially their biofilm microbial communities, are considered a valuable resource for novel antimicrobial molecules due to their ability to produce uniquely structured defensive metabolites.

[0003] Among the many marine microbial metabolites, the compound Alteramide A is a known example, with previous studies primarily revealing its antitumor cytotoxic activity. However, the development of this compound faces significant limitations: on the one hand, microbial strains capable of stably producing Alteramide A are extremely scarce; more importantly, current technologies focus entirely on its antitumor potential, while its potential antibacterial activity remains almost unknown, especially lacking quantifiable experimental evidence against common pathogens.

[0004] Therefore, discovering novel microbial strains capable of efficiently producing Alteramide A from unique marine habitats and systematically revealing and verifying their antibacterial activity and efficacy is of key significance for expanding the application boundaries of this compound and developing new antibacterial resources. Summary of the Invention

[0005] This invention aims to address the problem of scarce resources of Alteramide A-producing bacterial strains and the lack of sufficient verification of their antibacterial activity in existing technologies. This invention provides an alteramide A-producing strain, *Pseudomonas wenhaiensis* M044, and its applications. Experiments have for the first time demonstrated that this strain and its purified products exhibit significant inhibitory activity against *Staphylococcus aureus* and *Bacillus subtilis*, and its minimum inhibitory concentration (MIC) was determined. Simultaneously, genomic analysis successfully located its biosynthetic gene cluster.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a strain of *Pseudomonas wenhaiensis* M044 producing alteramide A and its applications, which was deposited on November 3, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20252408 and classification name [not specified]. Pseudoalteromonas maricaloris M044.

[0007] A second aspect of the present invention provides a microbial inoculum comprising the above-mentioned *Pseudomonas ventricosa* M044 and / or its ferments.

[0008] A third aspect of the present invention provides alteramide A, a metabolite produced by *Pseudomonas wenyusense* MO44 as described above, with the molecular formula C2. 29 H 38 N2O6.

[0009] A fourth aspect of the present invention provides an antibacterial composition comprising the above-described alteramide A and a pharmaceutically or agriculturally acceptable carrier.

[0010] The fifth aspect of the present invention provides the above-mentioned *Pseudomonas wenhaiensis* M044, or its use in the preparation of the above-mentioned formulation for inhibiting the growth of *Staphylococcus aureus* and / or *Bacillus subtilis*.

[0011] Furthermore, the preparation is an antibacterial drug, disinfectant, or preservative.

[0012] A sixth aspect of the present invention provides a method for producing the compound alteramide A, comprising culturing the strain described above in a culture medium containing seawater or inorganic salt components simulating seawater, and isolating and purifying alteramide A from the cultured species.

[0013] A seventh aspect of the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding an alteramide A biosynthetic enzyme system, said sequence being shown in SEQ ID NO:2.

[0014] An eighth aspect of the present invention provides a recombinant vector comprising the polynucleotides described above.

[0015] A ninth aspect of the present invention provides a genetically engineered host cell comprising the above-described polynucleotide or the above-described recombinant vector, wherein the host cell is capable of producing the compound alteramide A.

[0016] This invention provides a strain of *Pseudomonas maricaloris* M044 and its applications. The provided strain, *P. maricaloris* M044, can stably synthesize bioactive secondary metabolites such as alteramide A. Functional verification showed that this strain and its purified products exhibit significant inhibitory activity against *Staphylococcus aureus* and *Bacillus subtilis*, and their minimum inhibitory concentration (MIC) was determined. Furthermore, genomic analysis successfully located its biosynthetic gene cluster, indicating that this strain possesses a well-defined synthetic pathway and stable product synthesis capability. Compared with traditional antimicrobial compounds, the strain and its products of this invention are naturally derived, safe, and environmentally friendly, with a clear biosynthetic pathway, enabling efficient and controllable production processes. This provides a new microbial resource and technological approach for developing novel marine-derived antimicrobial agents. Attached Figure Description

[0017] Figure 1 The colony morphology is M044.

[0018] Figure 2 Phylogenetic tree of M044 and reference genome.

[0019] Figure 3 The results of the inhibition zone experiment of M044 bacterial suspension against four pathogenic bacteria are shown.

[0020] Figure 4 The results are the MS / MS detection results for compound alteramide A.

[0021] Figure 5 This is the structure of compound alteramide A.

[0022] Figure 6 The results of the inhibition zone experiment of compound alteramide A on four pathogenic bacteria are shown.

[0023] Figure 7 The results show the minimum inhibitory concentration (MIC) of compound alteramide A against four pathogenic bacteria.

[0024] Figure 8 This is the alteramide A biosynthesis gene cluster.

[0025] Figure 9 This is the result of Clinker alignment. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] The first aspect of this embodiment provides a strain of *Pseudomonas wenhynchophyton* M044, which was deposited at the China Center for Type Culture Collection (CCTCC) on November 3, 2025, with accession number CCTCC NO: M 20252408, and its classification name is... Pseudoalteromonas maricaloris M044.

[0029] The second aspect of this embodiment provides a microbial inoculum containing the aforementioned *Pseudomonas ventricosa* M044 and / or its ferments.

[0030] The third aspect of this embodiment provides a metabolite, alteramide A, produced by *Pseudomonas wenhaiensis* M044 as described above, with the molecular formula C. 29 H 38 N2O6.

[0031] The fourth aspect of this embodiment provides an antibacterial composition comprising the above-described alteramide A and a pharmaceutically or agriculturally acceptable carrier.

[0032] The fifth aspect of this embodiment provides the above-mentioned *Pseudomonas wenhaiensis* M044, or its use in the preparation of the above-mentioned formulation for inhibiting the growth of *Staphylococcus aureus* and / or *Bacillus subtilis*.

[0033] In some embodiments, the formulation is an antimicrobial agent, a disinfectant, or a preservative.

[0034] The sixth aspect of this embodiment provides a method for producing the compound alteramide A, comprising culturing the strain described above in a culture medium containing seawater or inorganic salt components simulating seawater, and isolating and purifying alteramide A from the cultured species.

[0035] The seventh aspect of this embodiment provides an isolated polynucleotide comprising a nucleotide sequence encoding an alteramide A biosynthetic enzyme system, the sequence being shown in SEQ ID NO:2.

[0036] The eighth aspect of this embodiment provides a recombinant vector comprising the aforementioned polynucleotides.

[0037] The ninth aspect of this embodiment provides a genetically engineered host cell containing the aforementioned polynucleotide or the aforementioned recombinant vector, and the host cell is capable of producing the compound alteramide A.

[0038] To better understand the technical solutions of the above embodiments, the following more detailed implementation examples are provided for further explanation.

[0039] Example 1: Isolation and genome sequencing of strain M044 Marine biofilms were collected from the surface of rocks in the subtidal zone of Maidong Island, Qingdao, Shandong Province, China (36°05' N, 120°43' E) using sterile swabs. The collected samples were suspended in sterile seawater and immediately transferred to the laboratory. After vortexing for 15 min, the bacterial cells were resuspended in sterile marine broth 2216E medium and diluted a series of gradients (10⁻⁶). 0 10 -1 10 -2 10 -3 10 -4 and 10 -5 Spread 100 μL of the diluted solution onto Marine 2216E agar plates and incubate at 25°C until visible colonies are formed. Subculture the resulting single colonies on Marine 2216E agar plates for at least 8 generations. Colony morphology should be as shown in the image. Figure 1 As shown, single colonies were then inoculated into 2216E liquid medium, and the bacterial culture was collected by centrifugation (6,000 rpm, 5 min) after 24 h of growth. After centrifugation, the supernatant was discarded, and the obtained bacterial pellet was used to extract genomic DNA. The 16S rRNA gene fragment of the DNA was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTTC-3'). The PCR products were Sanger sequenced at Sangon Biotech Co., Ltd. (Shanghai, China) to obtain the full-length 16S rRNA. Genomic libraries were prepared using the NEBNext Ultra DNA Library Prep Kit, and the concentration was determined using a Qubit 2.0 Fluorometer. The quality-compliant libraries were sequenced on the Illumina NovaSeq platform at the Novogene Institute of Bioinformatics in Beijing, yielding 2 Gb of sequencing data per strain. The genome draft of M044 was assembled using SPAdes software installed on a local Linux system. Quality assessment was performed using CheckM2, and species annotation of the genome was performed using GTDB-Tk.

[0040] Complete genomes of 19 previously reported Pseudoalteromonas species were downloaded from the NCBI website as references to construct a phylogenetic tree for M044. Thirty housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, and tsf) were extracted from the 20 genomes using AMPHORA2 software. The protein sequences corresponding to these housekeeping genes were aligned using MEGA7 software, and a phylogenetic tree was constructed in Jones-Taylor-Thornton mode using maximum likelihood (ML).

[0041] The phylogenetic tree results are as follows Figure 2 As shown, the results indicate that M044 is most closely phylogenetically distant from the species *Pseudoalteromonas maricaloris* SCSIO 43202. Based on species annotations from the GTDB database, strain M044 is classified as *P. maricaloris*, with the following taxonomic ranks: Domain: Bacteria; Phylum: *Pseudomonadota*; Class: *Gammaproteobacteria*; Order: *Enterobacterales*; Family: *Alteromonadaceae*; Genus: *Pseudoalteromonas*; Species: *Pseudoalteromonas maricaloris*. Combining the phylogenetic tree and species annotation results, M044 is identified as a novel strain of the species *Pseudoalteromonas maricaloris*. Strain M044 was deposited at the China Center for Type Culture Collection on November 3, 2025, with accession number CCTCC M 20252408.

[0042] Example 2: Detection of the inhibitory activity of M044 against pathogens The antibacterial activity of M044 was determined using the pore diffusion method. M044 was inoculated into fresh 2216E liquid medium and incubated statically at 25°C for 3 days. *Staphylococcus aureus* ATCC12600, *Bacillus subtilis* ATCC23857, *Staphylococcus aureus* ATCC33591, and *Staphylococcus aureus* ATCC33592 were inoculated into fresh 2216E liquid medium and incubated overnight at 37°C and 180 rpm. After autoclaving 2216E agar, the medium was cooled to approximately 50°C. 20 mL of the medium was then mixed with 10 μL of indicator strain seed culture and spread evenly in 90 mm diameter Petri dishes. After drying the plates, wells were created using a 5 mm punch. 50 μL of M044 bacterial suspension (with sterile 2216E liquid medium as a negative control) was added to each well. The plates were sealed and incubated upright at 25°C for 24 h. The size of the inhibition zone was then measured and photographed.

[0043] The inhibitory results of M044 on Staphylococcus aureus ATCC12600 and Bacillus subtilis ATCC23857 are as follows: Figure 3 As shown, the results indicated that strain M044 significantly inhibited Staphylococcus aureus ATCC12600, Bacillus subtilis ATCC23857, Staphylococcus aureus ATCC33591, and Staphylococcus aureus ATCC33592, with inhibition zone diameters of 1.8 cm, 2.2 cm, 1.7 cm, and 1.8 cm, respectively.

[0044] Example 3: Isolation and identification of the antibacterial compound alteramide A from M044 Strain M044 was plated on 2216E agar plates and incubated at 25°C for 7 days. After collecting the colony by scraping from the agar plate surface, methanol solution was added, and the mixture was ultrasonically extracted for 20 min in an ultrasonic cleaning tank. The extract was dried using a vacuum centrifuge, and the residue was resuspended in fresh methanol solution for 2 min using an ultrasonic cleaning tank. After centrifugation at 12,000 rpm for 10 min, the supernatant was collected to obtain the crude extract. The crude extract was filtered through a 0.22 μm nylon membrane filter and purified using an Agilent 1260 high-performance liquid chromatography system. The fraction from 13.9 to 14.5 min was collected, and the fraction was vacuum dried to obtain the pure compound. The compound was analyzed by HPLC-MS / MS using an Agilent 6530 LC / Q-TOF mass spectrometer and an Agilent 1290 ultra-high performance liquid chromatograph. Nuclear magnetic resonance (NMR) detection was performed using an Agilent DD2 500 spectrometer after dissolution in deuterated DMSO-d6.

[0045] The results showed that the isolated compound was a yellow powder with a molecular weight of 510.6 Da and a molecular formula of C2.29 H 38 N2O6, MS / MS detection results are as follows: Figure 4 As shown, the structure is as follows Figure 5 As shown, it has the same structure as the known compound alteramide A.

[0046] Example 4: Antibacterial activity analysis of compound alteramide A Staphylococcus aureus ATCC12600, Bacillus subtilis ATCC23857, Staphylococcus aureus ATCC33591, and Staphylococcus aureus ATCC33592 were inoculated into fresh MH liquid medium and incubated overnight at 37°C and 180 rpm. The indicator bacterial suspensions were evenly spread onto MH agar plates using the spread plate method. 20 μL of alteramide A solution dissolved in DMSO (with pure DMSO as a negative control) was added to a 0.6 cm sterile antimicrobial susceptibility test slide, which was then placed on the plate. The plate was sealed and incubated upright at 25°C for 24 h. The size of the inhibition zone was measured and photographed.

[0047] The inhibitory results of compound alteramide A on Staphylococcus aureus ATCC12600 and Bacillus subtilis ATCC23857 are as follows: Figure 6 As shown, the results indicated that strain M044 significantly inhibited Staphylococcus aureus ATCC12600, Bacillus subtilis ATCC23857, Staphylococcus aureus ATCC33591 and Staphylococcus aureus ATCC33592, with inhibition zone diameters of 1.1 cm, 1.2 cm, 1.0 cm and 1.1 cm, respectively.

[0048] Example 5: Determination of the minimum inhibitory concentration (MIC) of compound alteramide A against Staphylococcus aureus and Bacillus subtilis The MIC of compound alteramide A was determined using the broth microdilution method. Four pathogens were inoculated into sterile CAMHB liquid medium and incubated overnight at 37°C with shaking. Then, 1% of the pathogens were inoculated into fresh CAMHB liquid medium and cultured until the logarithmic growth phase. The bacterial concentration was then adjusted to 1 × 10⁻⁶. 5Bacterial suspensions were obtained at CFU / mL, and 180 μL of the suspension was transferred to 96-well plates. Alteramide A powder was dissolved in DMSO and diluted serially two-fold to obtain concentrations of 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, and 640 μg / mL. 20 μL of each concentration of alteramide A solution was added to the bacterial suspension in the 96-well plates, resulting in concentrations of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, and 64 μg / mL, respectively. After incubation at 37°C for 24 h, bacterial growth was assessed using a microplate reader. The MIC (minimum inhibitory concentration) was defined as the minimum alteramide A concentration at which no bacterial growth was detected. Sterile water served as a control group, and each experiment was performed in triplicate.

[0049] When determining the MIC of compound alteramide A against Staphylococcus aureus ATCC12600, Bacillus subtilis ATCC23857, Staphylococcus aureus ATCC33591, and Staphylococcus aureus ATCC33592, bacterial growth was observed as follows: Figure 7 As shown in the figure. The results showed that the MIC of compound alteramide A against Staphylococcus aureus ATCC12600 and Bacillus subtilis ATCC23857 was 8 μg / mL, the MIC against Staphylococcus aureus ATCC33591 was 16 μg / mL, and the MIC against Staphylococcus aureus ATCC33592 was 8 μg / mL.

[0050] Example 6: Analysis of biosynthetic gene clusters related to alteramide A synthesis in M044 Biosynthetic gene clusters in the M044 genome were predicted using the antiSMASH software under the default reference. Other biosynthetic gene clusters of polycyclic tetraamide macrolactone natural products were retrieved and downloaded, and then aligned with the reference gene clusters using Clinker software.

[0051] AntiSMASH prediction ( Figure 8 Align with Clinker ( Figure 9 The results collectively support that the M044 genome contains a PKS-NRPS type biosynthetic gene cluster related to alteramide A biosynthesis, and its core synthetic genes (sterol dehydrogenase, FAD-dependent oxidoreductase and NRPS-type I PKS fusion enzyme) are homologous to the previously reported PTM BGC in terms of sequence and module composition.

[0052]

[0053] The nucleotide sequence encoding the alteramide A biosynthetic enzyme system is shown in SEQ ID NO:2:

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Pseudomonas hygroscopicus* M044, characterized in that, It was deposited at the China Center for Type Culture Collection (CCTCC) on November 3, 2025, with accession number CCTCC NO: M 20252408, and its classification name is [not specified]. Pseudoalteromonas maricaloris M044.

2. A microbial inoculant, characterized in that, Contains *Pseudomonas hygroscopicus* M044 as described in claim 1, or a fermentation product of *Pseudomonas hygroscopicus* M044 as described in claim 1.

3. A metabolite, alteramide A, produced by *Pseudomonas ventricosa* MO44 as described in claim 1, characterized in that... The molecular formula is C 29 H 38 N2O6.

4. An antibacterial composition, characterized in that, It comprises the alteramide A as described in claim 3, and a pharmaceutically or agriculturally acceptable carrier.

5. The use of the *Pseudomonas wenhaiensis* M044 of claim 1, or the microbial agent of claim 2, or the alteramide A of claim 3, or the antimicrobial composition of claim 4, in the preparation of an agent for inhibiting the growth of *Staphylococcus aureus* or *Bacillus subtilis*.

6. The application according to claim 5, characterized in that, The preparation is an antibacterial drug, disinfectant, or preservative.

7. A method for producing compound alteramide A, characterized in that, This includes culturing *Pseudomonas urinaria* M044 as described in claim 1 in a culture medium containing seawater or simulated seawater inorganic salt components, and isolating and purifying alteramide A from the cultured species.

8. An isolated polynucleotide, characterized in that, It contains a nucleotide sequence encoding the alteramide A biosynthetic enzyme system, as shown in SEQ ID NO:

2.

9. A recombinant vector, characterized in that, It comprises the polynucleotide of claim 8.

10. A genetically engineered host cell, characterized in that, The host cell contains the polynucleotide of claim 8 or the recombinant vector of claim 9, and the host cell is capable of producing the compound alteramide A.

Images