Enhancing bovicin HC5 production in Streptococcus equinus HC5 through adaptive laboratory evolution under thermal stress

Abstract

Bovicin HC5, a bacteriocin produced by Streptococcus equinus HC5, demonstrates inhibitory activity against pathogenic and spoilage microorganisms. However, low production yields hinder its widespread application. This study investigated the impact of temperature on S. equinus HC5 growth and employed adaptive laboratory evolution (ALE) under heat stress to obtain variants with improved bovicin HC5 production. The optimal growth temperature for the wild-type strain was determined to be 42 °C, with growth ceasing above 49 °C. Following 400 generations of ALE at 47 °C and 48 °C, eight variants were selected. Two of these variants exhibited significantly enhanced bovicin HC5 production, reaching up to a 140% increase (P < 0.05). The variant with the highest bacteriocin yield showed increased expression of bvcA, the gene encoding the bovicin HC5 precursor peptide. This high-producing variant also displayed enhanced thermal resistance, a higher growth rate (μ = 1.33 ± 0.02 h^-1), and increased biomass accumulation (OD_600nm = 4.03 ± 0.06) at 48 °C compared to the wild-type strain (μ = 0.98 ± 0.04 h^-1; OD_600nm = 1.96 ± 0.12) (P < 0.05). Furthermore, the selected variants exhibited alterations in membrane composition, characterized by an increased concentration of saturated fatty acids and a reduced Zeta potential (P < 0.05). Genomic analysis of these variants identified mutations in genes involved in protein modification, transcriptional regulation, and cellular transport, including a lantibiotic permease. These results demonstrate the effectiveness of ALE for generating S. equinus HC5 variants with improved bovicin HC5 production and provide valuable insights for optimizing bacteriocin biosynthesis strategies. KEY POINTS: • The optimal growth temperature for the Streptococcus equinus HC5 strain was determined to be 42 °C, with growth ceasing above 49 °C • The variant Streptococcus equinus HC5 40048 with the highest bacteriocin yield showed increased expression of bvcA, the gene encoding the bovicin HC5 precursor peptide • ALE is an efficient metabolic engineering strategy to increase bacteriocin production in Streptococcus equinus HC5.

Keywords: Antimicrobial peptides; Comparative genomics; Natural selection.

Fig. 1

Adaptive laboratory evolution (ALE) workflow. A S. equinus HC5 was activated in PC medium at 42 °C for 24 h. B The culture was grown in PC agar in an anaerobic chamber for 48 h, and an isolated colony was selected and inoculated into anaerobic PC broth. C The ALE was conducted by serial transfers of the S. equinus HC5 inoculum (3% v/v, initial OD_600nm of 0.05) into Hungate tubes containing 5 ml of PC medium. The incubation temperature was set at 47 °C and was increased to 48 °C after 150 generations

Fig. 2

Effect of temperature on the growth of S. equinus HC5. A Arrhenius plot demonstrating the effect of temperature on the specific growth rate (µ, h⁻¹). The specific growth rate was determined through linear regression of the ln values of OD_600nm over time (h). The letter “a” denotes the highest mean among the treatments, as determined by the Tukey and/or Bonferroni test at a 95% significance level. B Effect of temperature on the OD_600nm of S. equinus HC5 after 24 h of cultivation. The presented values were subtracted from the initial OD_600nm (0.05). Identical letters indicate no significant difference according to Tukey’s test at a 95% significance level. Error bars represent the standard deviation

Fig. 3

Effect of ALE using heat stress on the growth of S. equinus HC5. A Effect of heat stress on the specific growth rate (μ, h⁻¹). B Effect of heat stress on OD_600nm. The ALE experiment was conducted at an incubation temperature of 47 °C for 150 generations, followed by 48 °C for 250 generations. Identical letters indicate no significant difference according to Tukey’s test at a 95% significance level. Error bars represent the standard deviation

Fig. 4

Growth kinetic parameters of wilt type S. equinus HC5 and the ALE-derived variants. A Maximum specific growth rate. B Optical density 600 nm (OD_600nm). C Dry mass (mg ml⁻¹). D pH values. Identical letters indicate no significant difference according to Tukey’s test at a 95% significance level. Error bars represent the standard deviation

Fig. 5

Efficiency of bovicin HC5 extraction methods at low temperature (4 °C for 16 h) and high temperature (100 °C for 10 min) from S. equinus HC5 cells. A Activity test of bovicin HC5 extracts obtained using low- and high-temperature extractions, with L. lactis ATCC 19345 as the indicator bacteria. Uppercase letters represent the identification of the extracts. B Identification of extracts corresponding to the respective S. equinus HC5 strains by uppercase letters and size of inhibition zones (mm) formed by bovicin HC5 activity. Identical lowercase letters indicate that the results did not differ significantly according to Tukey’s test at a 95% significance level

Fig. 6

Specific activity of bovicin HC5 extracts among different variants of S. equinus HC5. The strains were grown in anaerobic PC medium at 42 °C for 24 h. Bovicin HC5 was extracted at 100 °C for 10 min. The specific activity of bovicin HC5 was calculated by dividing the bacteriocin titer by biomass (AU ml⁻¹ mg dry cell mass⁻¹). Identical letters indicate no significant difference according to Tukey’s test at a 95% significance level. Error bars represent the standard deviation

Fig. 7

Expression of bovicin HC5 biosynthetic genes in the wild-type strain of S. equinus HC5 and variants selected by ALE. Cultures were grown at 42 °C, in anaerobic PC medium supplemented with 8 g/L. RNA extraction was performed after 12 h of fermentation (stationary phase). The ribosomal RNA 16S (16S rRNA) and DNA gyrase A (gyrA) genes of S. equinus HC5 were used as endogenous controls to calculate relative expression levels. Identical letters indicate no significant difference according to Tuke’s test at a 95% significance level. Error bars represent the standard deviation

Fig. 8

Bovicin HC5 biosynthesis pathway in S. equinus HC5 and summary scheme of genotypic/phenotypic changes in the HC5 40048 variant. The HC5 40048 variant shows increased expression of the bvcA gene (1) and higher production of bovicin HC5 (2). Genomic analyses revealed a mutation in the transporter permease, BvcE (3), which could prevent secretion of bovicin HC5 to the extracellular environment, contributing to the absence of bovicin HC5 activity in the cell-free supernatant (4). The variant HC5 40048 also showed an increase in saturated fatty acid concentration and membrane modifications (5), which contribute to greater affinity and accumulation of bovicin HC5 in the cell envelope (6). Taken together, these modifications alter the residual electrical charge in the cytoplasmic membrane (7)