Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production

Abstract

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCC^M2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G₈-52 genomes. The mutant strain G₈-52 (CCTCC^M2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L^-1 (0.65 g g^-1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L^-1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G₈-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G₈-52 produced a relatively stable ectoine yield (1.50 g L^-1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G₈-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G₈-52 is suitable for large-scale fermentation applications.

Keywords: Ectoine; Ectoine producer; Ectoine yield; Genomic analysis; Halomonas; UV mutagenesis.

Fig. 1

Schematic showing the experimental design with multiple rounds of ultraviolet mutagenesis for the wild-type H. campaniensis strain XH26

Fig. 2

Images showing colony morphology and scanning electron micrographs of wild-type strain XH26 and mutated strains. (A) Wild-type strain colony images. (A1–A3) Scanning electron micrographs of the wild-type strain at 3,000 × , 5,000 × , and 10,000 × magnification, respectively. (B, C) Colony morphologies of the mutated strains G₈-52 and strain G₈-44, respectively. (B1–B3, C1–C3) Scanning electron micrographs of G₈-52 and G₈-44 cells, respectively. Bars in the 3,000 × , 5,000 × , and 10,000 × microscopic images represent 5 μm, 5 μm, and 1 μm, respectively

Fig. 3

Growth curve and ectoine yield characteristics of the wild-type strain XH26 and the mutant strain G₈-52. A Ectoine standard curve based on HPLC analysis. B Ectoine accumulation by the wild-type strain XH26 in CMEA medium. HPLC was performed as follows: mobile phase of acetonitrile/water (4/1, v/v), detection wavelength of 210 nm, flow rate of 1.0 mL/min, column pressure of 3.48–4.76 MPa, column temperature of 30 °C, and detection amount of 10 µL. C Ectoine accumulation by the mutated strain G₈-52. D Growth of the mutated strain G₈-52 and subculture generations of mutated strains in CMEA medium. All strains were cultured in CMEA medium on a rotary shaker at 35 °C and with shaking at 120 rpm. Data represent means of triplicate values with error bars showing standard deviation