A method for producing melanin by a recombinant halomonas using acetic acid as a substrate

Abstract

The application provides a method for producing melanin by using acetic acid as a substrate by a recombinant halomonas, in particular, a method for producing melanin by using sodium acetate as a carbon source by halomonas and application thereof. The method builds an economic, sustainable, non-grain raw material and high-benefit melanin production platform, and plays an important role in further reducing production cost and improving sustainable development.

Description

Technical Field

[0001] This invention relates to the fields of fermentation and synthetic biology. More specifically, this invention relates to a method for producing melanin using *Halomonas*. Background Technology

[0002] Melanin is a general term for a class of heterogeneous polyphenolic or indole polymeric pigments widely found in the biological world. Based on differences in their biosynthetic precursors and chemical structures, melanin is generally classified into five major categories: eumelanin (dark brown), pheomelanin (yellowish-red), neuromelanin, allomelanin, and pyomelanin (Cao, W., Zhou, X., McCallum, NC, Hu, Z., Ni, QZ, Kapoor, U., Heil, CM, Cay, KS, Zand, T., Mantanona, AJ, et al. (2021). Unraveling the Structure and Function of Melanin through Synthesis. J. Am. Chem. Soc. 143, 2622-2637.). Eumelanin and pheomelanin are widely found in the skin, hair, and eyes of vertebrates, while isomelanin and pyomelanin are mainly found in microorganisms and plants (Wakamatsu, K., and Ito, S. (2023). Recent Advances in Characterization of Melanin Pigments in Biological Samples. International Journal of Molecular Sciences 24, 8305.). Based on their unique physicochemical properties, melanin shows broad application prospects in fields such as antioxidants, photoprotection, radiation shielding, heavy metal chelation, and bioelectronic materials.

[0003] Melanin is a special class of melanin with low or no nitrogen content. The concept was first proposed by Yabuuchi and Ohyama in 1972, referring to water-soluble brown pigments produced by certain microorganisms (Cao, W., Zhou, X., McCallum, NC, Hu, Z., Ni, QZ, Kapoor, U., Heil, CM, Cay, KS, Zand, T., Mantanona, AJ, et al. (2021). Unraveling the Structure and Function of Melanin through Synthesis. J. Am. Chem. Soc. 143, 2622-2637.). The biosynthetic pathway of pyo-melanin begins with the metabolism of tyrosine or phenylalanine: first, 4-hydroxyphenylpyruvate dioxygenase (hppD) catalyzes the conversion of 4-hydroxyphenylpyruvate to homogentisic acid (HGA); subsequently, homogentisic acid spontaneously oxidizes and polymerizes under alkaline conditions, ultimately forming pyo-melanin polymers (Zeng, Z., Guo, X.-P., Cai, X., Wang, P., Li, B., Yang, J.-L., and Wang, X. Pyomelanin from Pseudoalteromonas lipolyticareduces biofouling.). Unlike the highly cross-linked structures of eumelanin and pheomelanin, pyocyanin has a relatively regular linear polymer structure with a molecular weight typically in the range of 10-14 kDa. Its structure contains quinone units, endowing it with excellent electron transport capabilities and reversible redox activity (Turick, CE, Beliaev, AS, Zakrajsek, BA, Reardon, CL, Lowy, DA, Poppy, TE, Maloney, A., and Ekechukwu, AA (2009). The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1. FEMSMicrobiol Ecol 68, 223-235.). These properties give pyocyanin unique application potential in fields such as bioelectronic devices, antioxidants, and radiation protection materials.

[0004] Currently, the preparation of purulent melanin mainly relies on microbial fermentation. Studies have shown that various bacteria (such as...) can produce melanin. Shewanella oneidensis , Escherichia coli , Pseudoalteromonas lipolytica ) and fungi (such as Aspergillus fumigatus , Yarrowia lipolytica Both tyrosine and phenylalanine can synthesize pyomelanin in culture media containing tyrosine or phenylalanine. For example, by overexpressing the HPPD gene in *E. coli*, purpuric acid accumulation and the production of 315.5 mg / L of pyomelanin can be achieved (Seo, D., and Choi, K.-Y. (2020). Heterologous production of pyomelanin biopolymer using 4-hydroxyphenylpyruvate dioxygenase isolated from *Ralstonia pickettii* in *Escherichia coli*. *Biochemical Engineering Journal* 157, 107548.). However, these studies mostly focus on model strains or naturally occurring pyomelanin-producing strains, whose fermentation processes usually require strict sterilization conditions and have limited tolerance to extreme environments such as high salt content, thus limiting the low-cost, large-scale production of pyomelanin.

[0005] Meanwhile, significant progress has been made in the microbial synthesis of other types of melanin. For example, Chinese patent CN118325801A, using *E. coli* BL21(DE3) as the host, achieved eumelanin synthesis with tyrosine as a substrate by knocking out competing pathway genes and overexpressing tyrosinase genes, yielding 635 mg / L. Patent CN121555384A further modified the metabolic pathway, achieving an eumelanin yield of 18.5 g / L in a 5 L fermenter. However, these studies mainly utilize high-value carbon sources such as glucose and add tyrosine as a substrate to the culture medium, resulting in high costs.

[0006] Halomonas ( Halomonas spp *Halomonas* is a type of salt-tolerant or halophilic microorganism capable of open fermentation under high-salt, high-pH conditions. It possesses unique advantages such as strong resistance to contamination, low cultivation costs, and no need for sterilization, making it a promising next-generation industrial chassis strain attracting significant attention in synthetic biology and green biomanufacturing in recent years. *Halomonas* can efficiently utilize inexpensive carbon sources, and its genetic manipulation tools are becoming increasingly sophisticated, leading to its successful application in the biosynthesis of various high-value-added chemicals. However, to date, there are no reports on the metabolic pathways of melanin synthesis in *Halomonas*, thus a highly efficient purulent melanin synthesis system based on hyosclerotic acid from *Halomonas* has not yet been established.

[0007] Therefore, developing an engineered strain of Halomonas that can utilize inexpensive, non-grain carbon sources (such as acetic acid / sodium acetate), adapt to high-salt fermentation environments, and has a clear genetic manipulation background for the efficient production of purulent melanin is of significant research value and industrialization significance for overcoming the host limitations of existing technologies, expanding the industrial production boundaries of purulent melanin, and reducing fermentation costs. Summary of the Invention

[0008] In their carbon fixation research on evolved strains of *Haloxylon ammodendron* (using CO2 or its derivatives as a carbon source) (see Chinese Patent Application No. 2026105239350, filed on the same day as this application, entitled "A Method for Recombinant *Haloxylon ammodendron* to Produce Melanin Using CO2 and Acetic Acid as Substrates"), the inventors unexpectedly discovered through isotope tracing experiments that *Haloxylon ammodendron* can utilize acetic acid in the culture medium as an energy carrier, and the carbon flow can converge to aromatic amino acid metabolic pathways such as tyrosine metabolism, thereby generating melanin. In contrast, *Haloxylon ammodendron* cannot produce melanin when using glucose as a carbon source. Without limiting themselves to any theory, it is speculated that when the culture medium includes glucose as a carbon source, *Haloxylon ammodendron* is more likely to metabolize via glycolysis, preferentially synthesizing PHA, etc., while when using acetic acid (sodium) as a carbon source, *Haloxylon ammodendron* is more likely to enter the aromatic amino acid synthesis route via gluconeogenesis, thereby further generating melanin. The speculated melanin synthesis metabolic pathway in *Haloxylon ammodendron* is as follows: Figure 1 As shown.

[0009] Based on this newly discovered melanin synthesis metabolic pathway in *Halomonas*, this invention increases melanin precursors (e.g., by supplementing the culture medium with tyrosine) and knocks out their degradation genes. hmgA Or overexpress its synthetic gene hppD Furthermore, synthetic biology techniques such as knocking out bypass genes were used to further increase melanin production, and a method for producing melanin using acetic acid as a substrate by Halomonas was successfully established, thus completing this invention.

[0010] Therefore, one aspect of the present invention provides a method for producing melanin, the method comprising: fermenting and culturing Halomonas bacteria in a culture medium using acetic acid or acetate as a carbon source (e.g., as the sole carbon source); and extracting melanin from the fermentation broth.

[0011] In one specific embodiment, the melanin is an extracellular product.

[0012] In one specific embodiment, the halomonas is selected from... Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonassmyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (culture preservation number CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593).

[0013] In one specific embodiment, the acetate is an alkali metal acetate salt, preferably sodium acetate or potassium acetate, more preferably sodium acetate.

[0014] In a preferred embodiment, the culture medium contains 10 to 50 g / L (e.g., 10, 15, 20, 25, 30, 35, 40, 45 or 50 g / L) of sodium acetate.

[0015] In one specific embodiment, the *Haloxymonas* is a wild-type or recombinant *Haloxymonas*, preferably wild-type or recombinant. Halomonas bluephagenesis .

[0016] In one specific embodiment, the halomonas can be initially grown in an alkaline acetic acid medium (i.e., a medium that uses acetic acid or acetate as a carbon source (e.g., as the sole carbon source)) and the pH is adjusted by adding acetic acid.

[0017] In one specific embodiment, the *Halomonas* strain had the hyoscine 1,2-dioxygenase gene knocked out. hmgA ).

[0018] In a preferred embodiment, hmgA Gene knockout is achieved through CRISPR / Cas9 gene editing or homologous recombination.

[0019] In one specific embodiment, the homogentisate 1,2-dioxygenase (hmgA) belongs to EC:1.13.11.5, and preferably encodes the gene... hmgA .

[0020] In one specific embodiment, the gene hmgA The encoded amino acid sequence is shown in SEQ ID No. 1, and the gene sequence is shown in SEQ ID No. 2.

[0021] In one specific embodiment, the Halomonas strain expresses exogenous 4-hydroxyphenylpyruvate dioxygenase (hppD).

[0022] In one specific embodiment, the amino acid sequence of hppD is shown in SEQ ID No. 3, and its encoding gene sequence is shown in SEQ ID No. 4.

[0023] In one specific embodiment, the *Halomonas* strain had its polyhydroxyalkanoate (PHA) synthase gene knocked out. phaC ).

[0024] In one specific embodiment, the *Halomonas* strain also had the pyruvate kinase 1 gene knocked out. pyk1 ), branched acid mutase / prebenzoic acid dehydratase gene ( pheA ), and / or transcriptional regulatory factor tyrosine repressor protein genes ( tyrR ).

[0025] In a preferred embodiment, the gene knockout is achieved through CRISPR / Cas9 gene editing or homologous recombination.

[0026] In one specific embodiment, the *Halomonas* strain also overexpresses phosphoenolpyruvate carboxykinase (pck), 3-deoxy-D-arabinohepulose-7-phosphate synthase (aroG), and / or 4-hydroxyphenylpyruvate dioxygenase (hppD).

[0027] In one specific embodiment, the gene sequence of the pck is shown in SEQ ID No. 5.

[0028] In one specific embodiment, the gene sequence of aroG is shown in SEQ ID No. 6.

[0029] In one specific embodiment, the gene expression or overexpression described above employs an inducible promoter or a constitutive promoter, preferably selected from the Mmp1 promoter. luc promoter, lux promoter, lac promoter, trp promoter, tac promoter, araBAD promoter, fadBA promoter, cin promoter, cym promoter, sal promoter, van promoter, tet promoter, ttg promoter, phlf promoter, phaP The promoter, hypoxia-inducible promoter, bacterial quorum sensing-inducible promoter, temperature-sensitive promoter, pH-sensitive promoter, J23119 promoter, or combinations thereof, preferably the constitutive promoter is selected from the wild-type porin gene P.porin The promoter or a mutant thereof, wherein the mutant is selected from P porin1 P porin3 P porin42 P porin51 P porin58 P porin59 P porin68 P porin140 P porin141 P porin183 P porin192 P porin194 P porin203 P porin221 P porin226 P porin259 and P porin278 More preferably, the porin59 promoter or the porin140 promoter. These P porin The promoter and its mutants are disclosed, for example, in CN117143793B.

[0030] In one specific embodiment, the culture medium is supplemented with tyrosine.

[0031] In a preferred embodiment, tyrosine (e.g., 0.1 to 10 g / L, preferably 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L tyrosine) is added to the culture medium.

[0032] In summary, this invention provides a novel method for producing melanin using sodium acetate as a carbon source from *Halophilus* and its application. Experimental results show that the melanin concentration reached 10.07 g / L. This method establishes an economical, sustainable, and highly efficient platform for melanin production, playing a significant role in further reducing production costs and improving sustainable development. Attached Figure Description

[0033] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein: Figure 1 This invention demonstrates the first discovery of the melanin synthesis metabolic pathway in Halomonas; Figure 2 The study demonstrates that recombinant Halomonas bacteria can produce melanin through metabolic engineering. Figure 3 In Figure a, we show the strategy of metabolic engineering modification; in Figure b, we show the production of melanin using shake-flask fermentation experiments with the modified Halophilic bacteria (red indicates the knocked-out gene, and blue indicates the gene overexpressed on the endogenous plasmid). Figure 4To investigate the effect of tyrosine addition on melanin production in recombinant strains, the effect of adding 1 g / L tyrosine during shake-flask fermentation on melanin production in recombinant strains was shown. Figure 5 This study demonstrates the scale-up experiment of recombinant strains in a fermenter to investigate their melanin production capacity. Figure 6 Characterization spectrum of melanin in fermentation products; Figure 7 This demonstrates melanin production of TD01 in shake-flask medium containing 30 g / L sodium acetate as the sole carbon source, with or without the addition of 1 g / L tyrosine. Figure 8 This demonstrates melanin production in the recombinant strain of TD01; Figure 9 The CB39 plasmid map is shown; the plasmid backbone is derived from pHbPBC-phaAB. Re The pck, aroG, and hppD genes were expressed using the porin59 promoter; Figure 10 The CB40 plasmid map is shown; the plasmid backbone is derived from pHbPBC-phaAB. Re The pck, aroG, and hppD genes were expressed using the porin140 promoter.

[0034] Sequence List Description (Example sequences and sequences used in examples) SEQ ID No. 1 – hmgA Encoded amino acid sequence

[0035] MTEQLKYQNGFHNHFSSEALPGALPIGQNSPQKCAYGLYAEQLTGSAFTAPRHQNFRSWLYRIRPSVVQSAYQPLENNNVHTAPLDKPAADPNQMRWDPVSIPDAPTDFIDGLFTIAVNGDAGTQAGVGVHVYTFNEDMTERFFYNADGELLFVPQMGAIRLRTEFGELEIDNGEIAVIPRGVKFQVRKAQGVDAARGYICENYGSPLELPGLGPIGANGLANPRDFQSPVAAYEDLEGDYRLVAKFSGRFWETKLNHSPLDVVAWHGNCAPYKYNLANFNTINTVSFDHPDPSIFTVLTSPSDTPGMANLDFVIFPPRWMVAENTFRPPWFHRNLMSEFMGLIHGEYDAKAEGFTPGGASLHNSMSPHGPDAETFEKASNAELKPQFLGDTLAFMFESRYFFHPTPAALDADFRQRDYVDVWSTLRSHFNPNQP SEQ ID No. 2 – hmgA Gene sequence

[0036] SEQ ID No. 3 - Amino acid sequence of hppD

[0037] MGPFPHDAPKVAISDANPAGTDGFEFVEFAHPEPEKLDSLFRQMGFVPVAKHRDKSITVYRQGDINYLLNSEPNSHASAFIEAHGPCAPAMAWRVVDAQHALKRAVDLGAEEFTGNKSIDAPAVIGIGGSLLYFIDTYGEKGSCYSNEFEWLDEEDPKPKGFGFYYLDHLTHNVIRGNMDTWYKFYHDTFNFREIRYFDITGKVTGLTSRALTSPDGKIRIPINESADDHSQIEEYLREYNGEGIQHIAIATDDIYTGTDLIAAEGLEFMPGPPSIYYEKSLERVKDHQEPLDKLRNRGILIDGEGVVGGGETRILLQIFSKTVIGPIFFEFIQRKGDDGFGEGNFKALFESIEEDQINRGVLQSTAE SEQ ID No. 4 - hppD gene sequence

[0038] SEQ ID No. 5 – pck gene sequence

[0039] SEQ ID No. 6 – aroG gene sequence

[0040] SEQ ID No. 7 – Knockout phaC gRNA sequence tgaggatttgctgtcacgga SEQ ID No. 8 – Knockout pheA gRNA sequence gtcgcctacttaggccctga SEQ ID No. 9 – Knockout tyrR gRNA sequence ccggtattagaagccatccc SEQ ID No. 10 – porin59 promoter sequence ttgcgttcactggaatcccagtatagagtttgacctgcgagca SEQ ID No. 11 – porin140 promoter sequence ttgcgttcactggaatcccagtataagatttgacctgcgagca SEQ ID No. 12 – hmgA upstream homologous arm sequence

[0041] tcgcggtcaaacatctcgatgcgtacccggtcaccaaagcgcataaagggggttttcacctggccgtacagtatctgctcgaccatgcgcacttcggccaagcagctatagcctacgccaccgtcagccactggctttccagggccgccatcggggtctgggttggaaacagtgcccgagccaatcacagcgcccgcgccgagataacgggtcttggcggcatgggcaaccaactgagggaagctgaaaatcatatccggcccggcttctggctcgccaaacttctcgtcattcagatgcacggtcagcggcaggtgtaccttgccttcttgccaagcatcgcctagttcatcgggggtcaccgcgatgggcgagaaagaagaggcgggtttggcttggaaaaagccgaagcccttcgctagctcacctggaattaggccacgcaggctcacatcgttaaccagcattactagcttaatgtgcttggcggcttcctcgggagtgactgccatcggcacgtcatccgtaatgaccgcaatctcgccttcaaagtcgataccatgctcttcactcaccgcctcgatatcttcggtgggcgccaggaaacagtcgctaccgccctggtacatgagcggatcggtccagaaggtttcgggcatctcggcattgcgcgcctggcgtaccagctggacatggttcagataggcggagccgtcagcccactgataggcacgcggtaaaggagagtgcagggcacgctggtcaagctcaaaagcaccgtcggcatcgccaccgttcagttgtgcatagcgttcttcgagctgagggctaacggcttcccagctttcaagggccgcctgaagcgtgggagcaatatcaacagcgctcaccgcgcgggagaggtcacgggagacgatgataagttcgccgtcgcggcctttgttaagtgttgcaagtttcatggtcattcaatccttgtcgtcagggggcgataagtt SEQ ID No. 13 – hmgA downstream homologous arm sequence

[0042] SEQ ID No. 14 – Upstream primer of the upstream homologous arm of hmgA

[0043] atagggcccgatcccaagcttcttcgcggtcaaacatctcgatgc SEQ ID No. 15 – Downstream primer of the upstream homologous arm of hmgA

[0044] aacttatcgccccctgacgacaa SEQ ID No. 16 – upstream primer of the downstream homologous arm of hmgA

[0045] gtcgtcagggggcgataagtttgttaaggcctgctttatttgaacgtta Downstream primer of the downstream homologous arm of SEQ ID No. 17 – hmgA

[0046] ctcgatatcgcatgcggtaccaggccccaaaaacgcttttatcca SEQ ID No. 18 – Pyk1 upstream homologous arm sequence

[0047] tacacgcactgccaaaccatcgaacttacccgccagctccggcaataccaaaccaacggccgctgccgcgcctgttttggtcggaatcatcgagtgcgtcgcactacgcgcgcggtaagggtcggagtgataaacatccgataggttctgatcgttggtgtaagcatgaaccgtagtcatcaaaccattttcaatgcctacggcatcgtttaacgctttggcgaccggcgccaggcagttagtcgtgcacgacgcattggaaaccaccgtgtgctcggctgtcagaatgtcgtcattgacaccatatacgatggtggcgtcagcatcagggctgggcgcagagatcaacacacgcttagcacccgcctcaatatgcttggcagcagcctcacgcttggtaaacaggcctgtacactccatgacgaggtcaatgttcatggatgcccaaggaagttgactgggatcacgctcagaagaaattgcaatgcgatcgccatctacactgatactttcggcatcgtgttcaaccgcaaacgggaaatggccgtgaaccgtatcgtggcgtagcaggtgcgaattaagggaaggatcgcccagatcgttaatcgcaacgacttggacgcgatctcggtagctattttcgtacagcgcacgcagtacattgcggccaatccgtccaaatccgttaatggcaactcttattgtcatgatggcacctcaatcaagcagtggtagtagatggcgtatccagtaaaaaaattactaaaaatacgtatttattcacctttaggtcaatttatacctgtttcaaccgcccttaaaggctaataacgctccaatatgtagtaaaattactatataaacgctatcgtagtccaataattaatcaccgccaagacggtcaataggagtaacttttatgactcgctcaacatctcccaccccccttcgccgcacaaagattgttgcgacgcttggtccagcaagcgaccgtcctggt。 SEQ ID No. 19 – Pyk1 downstream homologous arm sequence

[0048] aactggcagaaacctgaaatgtgactgtgacctggagagccatgccatgaccgacgcacgcccaccccgccaagccgttcgtacgctgcaaccgcgtaagcgcattgcactgattgctcacgacggcaaaaaaaccgaaatgctggagtgggcaacgcgctggcaggaaacgctcagccagcacaccctaattggcacgggcacaacctctagcaagctgaagagcgccttagggctggaagttgaaggattgatgagcgggccactaggtggcgatcagcaaattggcgcgcgtattgctgagcagcagttggacgtgctgattttcttctgggatccgtttgcccctcagccccacgatcctgacgtcaaagcgctgctgcgcttagccgcattatggaacgtgccggtggcgtgcaatgcggcgagcgccgactttttactgtcttcgccctaccttagtgaacgctacgatatgtctattccggatgcagaagcctgggctcaggcccgcacggtctaaggccgcccgaccatatggttttaccgggtacgcaggtggcgcgccacgacctgctgcccgcactgatagtcaccgctcatggcattcagggcgcagtttagggtgagttcagcaatgcgatcataatcctgcagggctgaatttaccggttgcggcagaaaatccaacaagcggtggtcgccaaaggtggctaaccgcatctcttccggtagcccgccgcgttccagaaacacatcaaagaccccttccatcagtacataagacgcagtaaccagcgcatcagctccaccctcttcatccagcaattgggcagcaagacgagcgccttcgctacgctcatagtgcgtgccgctaagcaaaattggctcaatagggctgctttgcagggccgtctgaaagcccgtccgccgctcgcggctgatcgacaaacttggcatcgcctccagccacgccactcgccgcgtactatcagtaatg。 SEQ ID No. 20 – Upstream primer of the upstream homologous arm of Pyk1

[0049] tgatagggcccgatcccaagcttcttacacgcactgccaaac SEQ ID No. 21 – Downstream primer of the upstream homologous arm of Pyk1

[0050] aggtttctgccagttaccaggacggtcgctt SEQ ID No. 22 – Upstream primer of the downstream homologous arm of Pyk1

[0051] agcgaccgtcctggtaactggcagaaacctgaaatg SEQ ID No. 23 – Downstream primer of the downstream homologous arm of Pyk1

[0052] agagctcgatatcgcatgcggtacccattactgatagtacgcggc SEQ ID No. 24 – Gene Editing Expression hppD gRNA sequence used cgcagcacataatggtgctg Detailed Implementation

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Unless otherwise stated, the terms used herein have their general technical meanings as understood by those skilled in the art. For definitions and terms in this art, those skilled in the art are particularly recommended to refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Plainsview, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999).

[0055] In this invention, the singular articles “a” and “the” cover a plurality of indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by reference in their entirety.

[0056] The term "comprising" or "including" as used in this invention is an open-ended description, encompassing all specified components or steps described, as well as other specified components or steps that do not substantially affect the meaning; when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still possess the activity described in this invention.

[0057] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0058] In this invention, the term "Haloxymonas spp." Halomonas ")" includes but is not limited to Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Even better Halomonas bluephagenesis TD1.0 ( Halomonas bluephagenesis TD1.0 is in Halomonas bluephagenesis Inserted on the basis of TD01 lacI For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. et al. (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140), Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (CGMCC No. 19880) Halomonas campaniensisLS21 (CGMCC No. 6593) (All of the above strains have been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the Budapest Treaty and have been disclosed in previous patent applications. For example, CGMCC No. 4353 has been disclosed in CN102120973A, CGMCC No. 19880 has been disclosed in CN111593006A, and CGMCC No. 6593 has been disclosed in CN102925382A).

[0059] In this invention, the term "expression" can refer to "overexpression," which is defined as a gene expression level higher than the natural state, possibly achieved through increased transcription levels (producing more messenger mRNA) or improved translation efficiency (generating more functional proteins). In one specific embodiment, preferably, the expression or overexpression of the exogenous gene can be achieved by inserting the target gene into a non-translation site in the genome, or by plasmid overexpression. Preferably, gene insertion is performed using the CRISPR / Cas9 method. Preferably, plasmid overexpression is introduced into the chassis strain via electroporation or conjugation transformation. When the host is *Halomonas*, the plasmid vector can be a pSEVA series vector (Martinez-Garcia E...). et al. ,SEVA 2.0: an update of the Standard European Vector Architecture for de- / re-construction ofbacterial functionalities. Nucleic Acids Res 2015, 43 (Database issue): D1183-1189.) or toxin-antitoxin plasmid pHbPBC vector (Ren K, Zhao YQ, Chen GQ, et al. Construction of a Stable Expression System Based on the Endogenous hbpB / hbpC Toxin-Antitoxin System of Halomonas bluephagenesis). ACS Synth. Biol 2023, 13 (1): 61-67.

[0060] In this invention, the cultivation methods include, but are not limited to, seed culture, well plate culture, shake flask culture, or fermenter culture.

[0061] The culture can be continuous fermentation or fed-batch fermentation.

[0062] Preferably, the substrate is contained in a culture medium.

[0063] The culture medium can be solid, liquid, or semi-solid.

[0064] The culture medium can be an activation medium, seed medium, fermentation medium, fed-batch medium, fed-batch medium, etc.

[0065] Those skilled in the art can adjust the concentrations of the added substrate, nitrogen source, and inorganic salts as needed. The substrate includes a carbon source, CO2, and / or CO2 derivatives; the nitrogen source includes urea, aspartic acid, glutamine, peptone, etc.; and the inorganic salts maintain the osmotic pressure required by the microorganisms.

[0066] The carbon source includes, but is not limited to, one or more of glucose, gluconate, sodium acetate, sucrose acetate, xylose, or cellulose hydrolysate. Sodium acetate is preferred.

[0067] In one specific embodiment of the present invention, the substrate includes sodium acetate and / or bicarbonate.

[0068] Preferably, the concentration of the substrate in the culture medium is any value between 1 and 300 g / L, and more preferably any value between 30 and 100 g / L.

[0069] Preferably, the concentration of the carbon source in the culture medium is any value between 1 and 300 g / L, and more preferably any value between 30 and 100 g / L.

[0070] The nitrogen source concentration is any value between 0.001 and 100 g / L.

[0071] Preferably, the culture medium further includes inorganic salts, such as potassium salts and / or sodium salts. The potassium salts include, but are not limited to, one or more of potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium nitrate, or potassium dihydrogen phosphate. The sodium salts include, but are not limited to, one or more of sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, sodium nitrate, or sodium dihydrogen phosphate; preferably, the sodium salt is sodium chloride.

[0072] Preferably, the concentration of inorganic salts in the culture medium is any value between 0.0001 and 1 M.

[0073] When the CO2 derivative is inorganic, it can partially replace inorganic salts (such as NaCl) in the culture medium.

[0074] The following embodiments further illustrate the present invention in detail, but should not be construed as limiting the scope of the present invention or the specific methods described herein.

[0075] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0076] Unless otherwise specified, all reagents and materials used in the examples are commercially available.

[0077] Source of strains used in the examples: Halomonas bluephagenesis TD01 (CGMCC No. 4353) has been published in CN102120973A; Halomonas bluephagenesis TD1.0 is in Halomonas bluephagenesis Based on TD01 (CGMCC No. 4353), the following was inserted: lacI For strains obtained by gene (to facilitate IPTG-induced expression of the target gene), see Zhao, H. et al. (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140, and disclosed in granted Chinese patent applications Nos. 2021108133976, 2022113288365 and 2021100113917; the bacteria can be obtained from Tsinghua University.

[0078] The gene editing method used in the examples: For example, gene knockout or insertion methods use CRISPR / Cas9 gene editing technology; see the literature "CRISPR / Cas9 editing genome of extremophile". Halomonas spp. [J]. MetabolicEngineering, 2018, 47: 219-229. (Qin Q et al. 2018)".

[0079] The culture medium used in the examples includes: The shake flask culture media are shown in Table 1 below:

[0080] The fermentation tank culture medium is shown in Table 2 below:

[0081] The mother liquors used in the fermenters and shake flasks were the same. The content of each substance is referenced in the following article: Chen, Y.-L., Liu, X., Zhang, L.-Z., Yang, J.-S., Guo, W.-K., Zheng, S., Wang, J.-L., Wu, F.-Q., Yan, X., Wu, Q., et al. Cell Sizes Matter for Industrial Bioproduction, a Case of Polyhydroxybutyrate. Advanced Science, Volume 12, Issue 14, 2412256, 2025, https: / / doi.org / 10.1002 / advs.202412256.

[0082] Methods for extracting melanin (also refer to the method of Schmaler-Ripcke et al. (Schmaler-Ripcke, J. et al. (2009) Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in Aspergillus fumigatus. Applied and Environmental Microbiology 75, 493-503)): 1) After fermentation, the culture was centrifuged at 10,000 g for 5 minutes. The supernatant was acidified with 1M hydrochloric acid to adjust the pH to 2. The solution was then allowed to stand at room temperature for 48 hours, followed by centrifugation at 12,000 g for 15 minutes to precipitate melanin. After precipitation with deionized water, the precipitate was collected by centrifugation again and freeze-dried in a freeze dryer for 12 hours to obtain melanin powder for later use.

[0083] 2) Use ferric chloride to extract melanin from fermentation broth. Add ferric chloride powder to the culture supernatant after fermentation to make its concentration 10 g / L to precipitate melanin. The extraction operation after precipitation is the same as in 1).

[0084] Quantitative analysis: Melanin was quantified based on absorbance at 400 nm. A series of dilutions ranging from 0.1 to 5 g / L were prepared using self-extracted melanin standards, and a standard curve was established for quantitative calculation of melanin concentration in the fermentation broth.

[0085] Structural characterization: Melanin samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and analyzed using a 500 MHz nuclear magnetic resonance spectrometer (AVANCE NEO 500, Bruker). 1 H NMR spectral analysis (spectrals are shown in...) Figure 6 (b) In addition, FT-IR spectral analysis of the sample was performed using a Bruker Vertex 70 Fourier transform infrared spectrometer to further confirm its functional group structure (spectral images are shown in [reference]). Figure 6 (a)

[0086] The dry cell weight (CDW, g / L) mentioned in the examples is the ratio of the mass of the dried bacterial cells to the volume of the recovered bacterial solution.

[0087] Example 1: Recombinant Halomonas strain produces melanin using acetic acid In this experiment, *Halomonas* was used. Halomonas bluephagenesis Derivatives of TD01 (strain preservation number CGMCC4353) Halomonas bluephagenesis TD1.0 (abbreviated as TD1.0, see Zhao, H.) et al. (2017) Novel T7-1ike expression systems used for Halomonas. Metab Eng 39, 128-140) was used as the starting strain. To enable the strain to produce melanin exclusively, we used CRISPR / Cas9 gene editing technology to knock out the polyhydroxyalkanoate (PHA) synthesis gene on the basis of TD1.0. phaC (gRNA sequence is shown in SEQ ID No. 7), yielding TD1.0 Δ phaC A strain of bacteria, which is used as a chassis for melanin production.

[0088] In TD1.0 Δ phaC In this strain, to achieve melanin production, we blocked the degradation of homogentisic acid by knocking out the hmgA gene (construction of the knockout plasmid: knockout was performed using a suicide plasmid method with pRE112-6I). -Sce I as a template expression hmgA Homologous arm (homologous arm sequences and amplification primers are described in the sequence listing above), pRE112-6I -Sce For details on plasmid and homologous recombination methods, please refer to Fu et al., Development of HalomonasTD01 as a host for open production of chemicals, Metabolic Engineering, volume 23, May 2014, pages 78-91, 2014 (see also CN105779488B), to obtain recombinant strain TD1.0 Δ phaC Δ hmgA This strain, when fermented in shake flasks with sodium acetate (30 g / L) as the sole carbon source, produced 0.20 g / L of melanin (identification chromatogram shown). Figure 6 (As shown). Based on this, to increase melanin production, we used CRISPR / Cas9 gene editing technology to insert the 4-hydroxyphenylpyruvate dioxygenase gene expressed by the porin59 promoter (SEQ ID No. 10) at the G58 site of the genome. hppD (SEQ ID No. 4) (gRNA sequence used for gene editing: cgcagcacataatggtgctg (SEQ ID No. 24)), strain TD1.0 Δ phaC Δ hmgA-hppD Melanin production increased to 1.14 g / L ( Figure 2 ).

[0089] Example 2: Metabolic engineering to further increase melanin production To further increase melanin production, the melanin synthesis pathway was studied. Figure 1 We plan to block TD1.0 ∆ phaC ∆ hmgA-hppD The strain's melanin synthesis pathway genes and transcriptional regulatory factors were sequentially knocked out, with pyruvate kinase 1 (Pkinase 1) being eliminated. pyk1 Construction of the knockout plasmid: Knockout was performed using the suicide plasmid method with pRE112-6I. -Sce I is used as a template to express the homologous arm of pyk1 (the homologous arm sequence and amplification primers are described in the sequence listing section above), pRE112-6I - Sce For details on plasmid and homologous recombination methods, please refer to Fu et al., Development of HalomonasTD01 as a host for open production of chemicals, Metabolic Engineering, volume 23, May 2014, pages 78-91, 2014, also see CN105779488B), branched acid mutase / prephenyl acid dehydratase (pheA; CRISPR / Cas9 method, gRNA knockout: gtcgcctacttaggccctga (SEQ ID No. 8)), and transcriptional regulator tyrosine repressor protein (tyrR; CRISPR / Cas9 method, gRNA knockout: ccggtattagaagccatccc (SEQ ID No. 9)), to obtain strain DJ115.

[0090] Because *Halomonas* possesses endogenous plasmids, we used an endogenous plasmid system to enhance the metabolic flux of melanin. In the melanin synthesis pathway, the three key rate-limiting enzymes are phosphoenolpyruvate carboxykinase (pck), 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroG), and 4-hydroxyphenylpyruvate dioxygenase (hppD). Therefore, we used an endogenous plasmid system to express these three genes using the porin59 promoter (ttgcgttcactggaatcccagtatagagtttgac (SEQ ID No. 10)) and the porin140 promoter (ttgcgttcactggaatcccagtataagatttgacctgcgagca (SEQ ID No. 11)), obtaining two plasmids, CB39 and CB40 (plasmid maps are shown below). Figure 9 and Figure 10The plasmid backbones were all derived from pHbPBC-phaABRe, from, for example, the literature: Chen, Y.-L., Liu, X., Zhang, L.-Z., Yang, J.-S., Guo, W.-K., Zheng, S., Wang, J.-L., Wu, F.-Q., Yan, X., Wu, Q., et al. Cell Sizes Matter for Industrial Bioproduction, a Case of Polyhydroxybutyrate. Advanced Science, Volume 12, Issue 14, 2412256, 2025, https: / / doi.org / 10.1002 / advs.202412256) in order to increase the melanin synthesis capacity of the DJ115 strain. Plasmids CB39 and CB40 were transformed into strain DJ115, resulting in two strains, DJ116 (DJ115 carrying plasmid CB39) and DJ117 (DJ115 carrying plasmid CB40). Shake-flask experiments revealed... Figure 3 There was no significant difference in melanin production between strains DJ115 and TD1.0 ∆phaC ∆hmgA-hppD. However, strain DJ116 produced 1.35 g / L of melanin, which was higher than that of DJ115.

[0091] To enhance precursor supply, we added 1 g / L L-tyrosine to the shake-flask culture medium of strain DJ115 to increase melanin production. Shake-flask fermentation showed ( Figure 4 Melanin production was significantly increased compared to the group without the addition, from 1.14 g / L to 6.74 g / L.

[0092] Example 3: Fermentation in a 7-liter bioreactor To further verify the industrialization potential of the recombinant strain, fed-batch fermentation of strain DJ116 was conducted in a 7-liter fermenter using sodium acetate as the carbon source. Dissolved oxygen was controlled at 30%, and the pH was adjusted to around 9 using acetic acid. Considering that the initial addition of tyrosine would increase melanin production, two fermenters were designed: one with 1 g / L L-tyrosine and the other without. Fermentation results showed (see...) Figure 5 Without the addition of tyrosine, strain DJ116 fermented for 96 hours, yielding 4.15 g / L of melanin and OD... 600nm The OD value was 10.07. With the addition of 1 g / L tyrosine, strain DJ116 fermented for 96 hours, achieving a melanin yield of 10.07 g / L. 600nm It is 12.

[0093] In summary, we have achieved melanin production for the first time using acetic acid as the sole carbon source. With the supplementation of precursor tyrosine, the melanin yield was 10.07 g / L. Without the supplementation of precursor tyrosine, the yield of melanin synthesized de novo using acetic acid reached 4.15 g / L.

[0094] Example 4: Halomonas Halomonas bluephagenesis Experiments in TD01 In this experiment, *Halomonas* was used. Halomonas bluephagenesis Using TD01 (strain accession number CGMCC4353) as the starting strain, melanin production experiments were conducted in shake-flask media containing 30 g / L sodium acetate as the sole carbon source, with and without the addition of 1 g / L tyrosine. The results are as follows: Figure 7 As shown (TCM (True cell mass) represents the true cell mass). Surprisingly, even without gene recombination, wild-type TD01, using acetic acid as the sole carbon source, was still able to produce a small amount of melanin and synthesize polyhydroxybutyrate (PHB) under conditions of added tyrosine, while simultaneously synthesizing PHB (see CN120082497A for methods of measuring PHB). In contrast, under the same experimental conditions using glucose as the carbon source, *Halomonas TD01* could not produce melanin.

[0095] Next, similar to the gene editing method for TD1.0 described in the above embodiments, knockout was achieved in TD01. hmgA strain TD01∆ of the gene hmgA and knockout hmgA Genes and overexpression hppD strain TD01∆ hmgA-hppD Melanin production experiments were conducted in shake-flask culture medium containing 30 g / L sodium acetate as the sole carbon source under conditions without the addition of tyrosine. The results are as follows: Figure 8 As shown, based on the newly discovered melanin metabolism pathway in *Halomonas*, similar to TD1.0, the above gene recombination operation can further significantly increase melanin production in *Halomonas* TD01.

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0097] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. Furthermore, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, and should also be regarded as the content disclosed by the present invention.

Claims

1. A method for producing melanin, the method comprising: Halomonas bacteria are cultured by fermentation in a medium that uses acetic acid or acetate as a carbon source. And extracting melanin from fermentation broth.

2. The method according to claim 1, wherein the halomonas is selected from... Halomonas bluephagenesis , Halomonas aydingkolgenesis, Halomonas campaniensis , Halomonas lutescens , Halomonas hydrothermalis , Halomonas sp. KM1、 Halomonas elongata and Halomonas smyrnensis Preferred Halomonas bluephagenesis TD1.0 Halomonas bluephagenesis TD01 (CGMCC No. 4353) Halomonas aydingkolgenesis M1 (culture preservation number CGMCC No. 19880) and Halomonas campaniensis LS21 (CGMCC No. 6593).

3. The method according to claim 1 or 2, wherein the acetate is an alkali metal acetate, preferably sodium acetate or potassium acetate, more preferably sodium acetate.

4. The method according to any one of claims 1 to 3, wherein the halomonas is wild-type or recombinant halomonas, preferably wild-type or recombinant. Halomonas bluephagenesis It can be grown in an alkaline acetic acid medium, with the pH adjusted by adding acetic acid.

5. The method according to any one of claims 1 to 4, wherein the halomonas has had the hyoscine 1,2-dioxygenase gene knocked out. hmgA ).

6. The method according to any one of claims 1 to 5, wherein the Halomonas expresses exogenous 4-hydroxyphenylpyruvate dioxygenase (hppD).

7. The method according to any one of claims 1 to 6, wherein the Halomonas strain has had its polyhydroxyalkanoate (PHA) synthase gene knocked out. phaC ).

8. The method according to any one of claims 1 to 7, wherein the *Halomonas* strain has also had the pyruvate kinase 1 gene knocked out. pyk1 ), branched acid mutase / prebenzoic acid dehydratase gene ( pheA ), and / or transcriptional regulatory factor tyrosine repressor protein genes ( tyrR ).

9. The method according to any one of claims 1 to 8, wherein the *Haloxymonas* strain further overexpresses phosphoenolpyruvate carboxykinase (pck) and 3-deoxy-D-arabinohepulose-7-phosphate synthase (aroG), preferably overexpressing pck, aroG and hppD.

10. The method according to any one of claims 1 to 9, wherein the culture medium is supplemented with tyrosine.

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