Characterization and Biosynthetic Regulation of Isoflavone Genistein in Deep-Sea Actinomycetes Microbacterium sp. B1075

Abstract

Deep-sea environments, as relatively unexplored extremes within the Earth's biosphere, exhibit notable distinctions from terrestrial habitats. To thrive in these extreme conditions, deep-sea actinomycetes have evolved unique biochemical metabolisms and physiological capabilities to ensure their survival in this niche. In this study, five actinomycetes strains were isolated and identified from the Mariana Trench via the culture-dependent method and 16S rRNA sequencing approach. The antimicrobial activity of Microbacterium sp. B1075 was found to be the most potent, and therefore, it was selected as the target strain. Molecular networking analysis via the Global Natural Products Social Molecular Networking (GNPS) platform identified 25 flavonoid compounds as flavonoid secondary metabolites. Among these, genistein was purified and identified as a bioactive compound with significant antibacterial activity. The complete synthesis pathway for genistein was proposed within strain B1075 based on whole-genome sequencing data, with the key gene being CHS (encoding chalcone synthase). The expression of the gene CHS was significantly regulated by high hydrostatic pressure, with a consequent impact on the production of flavonoid compounds in strain B1075, revealing the relationship between actinomycetes' synthesis of flavonoid-like secondary metabolites and their adaptation to high-pressure environments at the molecular level. These results not only expand our understanding of deep-sea microorganisms but also hold promise for providing valuable insights into the development of novel pharmaceuticals in the field of biopharmaceuticals.

Keywords: biological activity; biosynthetic genes; deep-sea actinomycetes; flavonoid compounds.

Figure 1

Identifying Microbacterium sp. B1075 as the target strain. (a) The antibacterial activity of strain B1075 was validated using the K-B paper disk method, with methanol solution added in the blank control group. (b) The inhibition zones were measured in two diameters, and the corresponding inhibition values were obtained by taking the average. (c) Neighbor-joining tree based on the 16S rRNA gene sequences of Microbacterium sp. B1075 and closely related taxa within the Microbacterium genus. (d) Macroscopic image displaying the colony morphology of Microbacterium sp. B1075. (e) Growth rates of strain B1075 at different temperatures (n = 3). (f) Growth rates of strain B1075 at different salinities (n = 3). (g) Growth rates of strain B1075 at different pH values (n = 3).

Figure 2

Genome annotation of strain B1075. (a) The circular map depicts the CDSs on the positive and negative strands, with different colors indicating different COG functional classifications. The first and fourth circles represent CDSs on the positive and negative strands, respectively, while the second and third circles represent tRNA and rRNA. The fifth circle represents the GC content, the sixth circle represents the GC-Skew value, and the innermost circle indicates the genome size (source: cloud.majorbio.com). (b) Pie chart showing the percentage of metabolism-related genes annotated using the COG database for strain B1075. (c) Pie chart illustrating the percentage of metabolism-related genes annotated using the KEGG database for strain B1075.

Figure 3

Annotation results of secondary metabolites produced by strain B1075 using GNPS. (a) HPLC chromatogram of Fr-2 fraction of secondary metabolites isolated from strain B1075 after separation by ODS column. (b) Number of known compounds annotated by GNPS for secondary metabolites Fr-2 produced by strain B1075. (c) Molecular network analysis of secondary metabolites Fr-2 produced by strain B1075 using LC-MS/MS technology in positive and negative ion modes. Red nodes represent annotated known compounds, while blue nodes represent unknown compounds. (d) Identification of 25 flavonoid compounds (Table S2) annotated by GNPS for Fr-2 fraction.

Figure 4

Isolation and identification of secondary metabolites and antibacterial activity of strain B1075. (a) Measurement of the diameter of inhibition zones of three fractions of secondary metabolites from strain B1075 separated by an ODS column. Fractions: 0–35 represents Fr-1; 35–65 represents Fr-2; 65–80 represents Fr-3; Control represents ‘control’ group (methanol). (b) Chromatogram of compound genistein obtained after the purification of secondary metabolite Fr-2 by HPLC. (c) Chemical shifts of carbon and hydrogen spectra along with the molecular structure of the isolated compounds. (d) Antibacterial activity values of compound genistein measured using the microdilution method.

Figure 5

Pathway for the synthesis of flavonoid compounds in strain B1075. (a) Enzyme genes in strain B1075 predicted to be associated with the biosynthetic pathway are highlighted in red. Enzyme gene with sequence similarity below 30% is displayed in black, and the catalytic step is indicated by dashed line. TAL, tyrosine ammonia-lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase. (b) Homologous sequences of CHS with similarity exceeding 60% were obtained by amino acid sequence alignment of strain B1075 through NCBI. Among them, the amino acid sequence similarity of GAT73531.1 reached 82.94%, represented by a gradient from yellow to blue, with higher similarity shown in yellow and lower similarity in blue. Protein domain prediction of the CHS fragment (encoded by the gene CHS) in strain B1075 was conducted using InterPro. Blue bar represents the N-terminal domain of chalcone and stilbene synthases identified in CHS of B1075. Yellow bar represents the C-terminal domain of chalcone and stilbene synthases identified in CHS of B1075. Graphical representation was generated using Jalview 2.11.3.0.

Figure 6

Impact of high hydrostatic pressure on the production of flavonoid compounds by strain B1075. (a) HPLC chromatograms of secondary metabolites extracted from strain B1075 under different hydrostatic pressure conditions. (b) Peak areas of genistein produced by strain B1075 at different pressures. The peak area at 60 MPa is approximately twice that at 0.1 MPa. Data represent the mean values of three independent experiments with similar results. (c) Relative mRNA expression level of the gene CHS under different pressure conditions. Error bars indicate the standard deviations from three independent experiments (*** p < 0.001, ** p < 0.01).