The strains and plasmids used in this study and their references are summarized in Table 1.

Escherichia coli DH5α was grown at 37°C in Luria–Bertani (LB) broth with vigorous agitation (220 rpm). Lactococcus lactis NZ9000 was grown at 30°C in GM17 broth containing 0.5% glucose (m/v). Clostridium beijerinckii ATCC 25752 and C. perfringens were grown anaerobically at 37°C in Reinforced Clostridial Medium (RCM). Lactobacillus sake ATCC 15521 was grown at 30°C in De Man, Rogosa, and Sharpe broth (MRS). Minimum Essential Medium (MEM) (Rink et al., 2005) was used whenever cell culture supernatant was subjected to peptide purification. For cell growth on plates, 1.5% agar was added to the corresponding broth. When required, ampicillin and chloramphenicol were used for E. coli DH5α at 100 μg/ml and 10 μg/ml, respectively. Chloramphenicol and erythromycin were used at 5 μg/ml each for L. lactis NZ9000. All the media and reagents mentioned above were purchased from Sigma-Aldrich unless stated otherwise.

The techniques of standard molecular cloning were performed as previously reported (Green et al., 2012). Genomic DNA from C. beijerinckii ATCC 25752 was extracted using the GenElute genomic DNA kit (Sigma-Aldrich, Zwijndrecht, Netherlands) and subjected to DNA amplification. The primers used for cloning were ordered from Biolegio (Nijmegen, Netherlands) (Supplementary Table 1). The NucleoSpin gel and PCR cleanup kit (BIOKÉ, Leiden, Netherlands) and the NucleoSpin Plasmid EasyPure kit (BIOKÉ, Leiden, Netherlands) were applied to purify PCR fragments and isolate plasmids, respectively. The obtained PCR products were ligated using Gibson Assembly Master Mix (BIOKÉ, Leiden, Netherlands) or USER Enzyme (NEB, Leiden, Netherlands) according to the manufacturers’ instructions. To achieve a high transformation efficiency, E. coli DH5α was used as an intermediate host for various constructs using the vector of pMG36c or pTLR4. The constructs using pNZ8048 vector were transformed directly into L. lactis NZ9000 competent cells by electroporation with the following settings: 2.25 kV, 25 μF, 200 Ω (Gene Pulser, Bio-Rad, Lunteren, Netherlands). The successful incorporation of desired mutations was verified by gene sequencing (Macrogen Europe, Amsterdam, Netherlands). The constructed L. lactis strains were stored at −80°C for further experiments.

For expression of circularin A, the heterologous host strain L. lactis was first inoculated at normal GM17 medium and grown overnight at 30°C. The next day, 2 ml fresh overnight culture was transferred into 100 ml of MEM supplemented with 0.5% glucose. The cells were grown at 30°C until the OD600 reached 0.5, then nisin was added at a final concentration of 5 ng/ml to induce bacteriocin production. After induction at 30°C for 16–20 h, the cell culture was centrifuged (8000 g, 15 min) and supernatant was collected for subsequent purification.

To purify circularin A, both methods of Trichloroacetic acid (TCA) precipitation and C18 purification were performed. The protocol for TCA precipitation was similar as described previously (Koontz, 2014). For C18 purification, 0.3 g C18 material (Sigma-Aldrich, Zwijndrecht, Netherlands) was packed in an open column and pre-washed with a solution consisting of 50% solution A (acetonitrile: isopropanol = 1:1, containing 0.2% TFA) and 50% solution B (milliQ water containing 0.2% TFA), then equilibrated with solution B. After column equilibration with 5 column volumes (CV), 100 ml collected supernatant was passed through the column by gravity, followed by a two-step wash and two-step elution process, which only differed in concentrations of solution A. The solutions applied to the washing steps were 20 and 50% solution A, respectively. The solutions used for the elution steps were 80 and 100% solution A, respectively. Each fraction was collected for 8 ml (around 10 column volume), freeze-dried (FreeZone, Labconco, Kansas City, MO, USA), then dissolved in 100 μl of 50% solution A, from which 1 μl sample was analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) as previously described (Zhao et al., 2020). The rest of the peptide was stored at 4°C for further analysis such as protein gel and activity assay.

The production levels of the circularin A and its mutants were assessed by tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (tricine-SDS-PAGE) using a 16% running gel and a 4% stacking gel. The gels were prepared as described previously (Schägger, 2006). The amount of each peptide subjected to the gel was purified from 20 ml cell culture (1/5 of the purified peptide from the previous step). The C18 elution fractions were first freeze-dried and then dissolved in 20 μl of milliQ water. 5 μl of the loading buffer (10% SDS, 0.5% Bromophenol blue, 50% glycerol, 250 mM Tris–HCl, pH6.8) was added into the peptide sample, and the mixture was heated at 50°C for 30 min. 5 μl of the PageRuler Protein Ladder (Thermo Fisher, Groningen, Netherlands) was used as a protein molecular weight marker and run alongside the peptide samples. After separation, peptide bands were visualized by staining in 0.2% (m/v) solution of Coomassie Brilliant Blue R-250 (Bio-Rad, Merck, Darmstadt, Germany) for 20 min and subsequent destaining in a solution containing 10% acetate acid and 20% ethanol (v/v). After gel destaining, peptide yields were visualized and estimated by band intensities in Image Lab 3.0 (Bio-Rad Laboratories, Hercules, CA, USA).

To achieve a better production of the antimicrobial peptide, various parameters were examined with a strain of L. Lactis containing the constructed plasmid pTLR4-Cir (Em^r, erythromycin resistance). The expression and purification procedures of circularin A were similar as described above. Briefly, the “standard condition” prior to optimization was used as follows: the cells were grown in the MEM supplemented with 0.5% glucose, 1% glycerol, 5 μg/ml erythromycin; when the cell growth reached the stage of OD600 ∼ 0.5, 10 ng/ml nisin was added to the cell culture to induce bacteriocin production at 30°C for 16–20 h. The antimicrobial production under this condition was set as 100% and used for comparison to evaluate peptide production under conditions of the altered parameters. The optimized parameters were mainly studied on the effect of the modification of the growth medium, such as the amount of carbon source, the effect of glycerol and antibiotic. Moreover, the influence of nisin-induction at different cell growth phases was also investigated. Compared with the above “standard condition,” five expression conditions, each with one changed parameter, were implemented as follows: (1) nisin-induction from the start-point of the expression incubation; (2) additional 50 mm sucrose (∼1.7%, w/v) in growth medium; (3) additional 1.7% glucose in growth medium; (4) absence of erythromycin in growth medium; (5) absence of glycerol in growth medium. The effect of each parameter was investigated independently and the peptide production was evaluated with a scale of production level as 0–10% (±), 10–50% (+), 50–150% (++), 150–500% (+++). A negative control strain lacking the peptide precursor gene (ΔcirA) was also included in this experiment.

To facilitate the investigation of circularin A mutants, site-directed mutagenesis was performed with the two-plasmid expression system (Cir-A +ΔA). In this system, primers were designed using the inverse PCR technique to insert desired mutations into cirA, while the rest of the circularin A gene cluster remained intact. The experimental procedures of DNA amplification, plasmid construction, and transformation were the same as described above. Finally, the correctly sequenced cirA mutants were transformed by electroporation into L. lactis NZ9000 harboring the rest of the biosynthetic proteins (cirΔA) as described earlier. All the primers used in this study are listed in Supplementary Table 1.

Antimicrobial activity of strains was assessed by colony overlay assays. Specifically, GSM17 medium (containing 0.5% glucose and 0.5M sucrose) supplemented with 5 ng/ml nisin and 1.5% (w/v) agar, served as the first layer (bottom layer). 2 μl overnight culture producer strain was spotted on the bottom layer and incubated at 30°C for 4–6 h before the second layer (top layer) seeded with an indicator strain was poured on top. The indicator strains used in this study were L. sake ATCC 15521, C. perfringens and L. lactis NZ9000, respectively.

When L. sake ATCC 15521 was used as the indicator strain, it was firstly inoculated in MRS liquid medium and cultivated at 30°C for 2 days, then the obtained cell culture was diluted 1,000-fold into MRS medium supplemented with 1% agar (the second layer). When L. lactis NZ9000 was used as the indicator strain, it was firstly inoculated in GM17 liquid medium and grown overnight at 30°C, then the overnight cell culture was diluted 10,000-fold into GM17 medium supplemented with 1% agar (the second layer). When C. perfringens was used as the indicator strain, it was firstly grown anaerobically in RCM liquid medium at 37°C for 24–36 h, and then the obtained cell culture was diluted 1,000-fold into RCM supplemented with 1% agar (the second layer). Ultimately, these two-layer testing plates were incubated at 30°C for 36–48 h in the case of L. sake ATCC 15521 and 16–20 h in the case of L. lactis NZ9000. The C. perfringens two-layer testing plate was grown in an anaerobic chamber at 37°C for 24–36 h.

The aa sequence of circularin A (GenBank: AAN86036.1) was aligned with that of enterocin AS-48 (GenBank: AHL69645.1), the prototype of circular bacteriocins. The candidate sequences were obtained from the NCBI database and aligned using Clustal Omega.¹ The alignment result was viewed and edited with Jalview version 2.11.2.1 (Geoff Barton’s group, University of Dundee).

The three-dimensional (3-D) peptide structure was modeled by input of the aa sequence of circularin A in the SWISS-MODEL website,² then the PDB formatted file containing the structural information could be downloaded and edited in PyMol.³

To achieve functional production of circularin A in L. lactis, various approaches were applied (Figure 2A): (1) cirB was separately put under the nisin-inducible promoter in the pNZ8048e plasmid, while the rest of the genes in the cirABCDE gene cluster were placed in pMG36c behind a constitutive promoter p32; (2) all five genes in the cirABCDE gene cluster were cloned into one single plasmid behind the nisin inducible promoter, creating pTLR4-CirABCDE, as well as (3) a variant form of the two-plasmid system (pNZ-CirA and pTLR4-CirΔA) was constructed to facilitate the later investigation of circularin A variants. Eventually, three different formats for cloning the cirABCDE gene cluster into L. lactis were completed, which included either one-plasmid approach pTLR4-CirABCDE, or the complementary constructs pNZ-CirA and pTLR4-CirΔA, and pMG-CirΔB and pNZ-CirB. The complementary design facilitates further research, especially on mutagenesis studies by creating an efficient cloning and production system to obtain sufficient amounts of various modified mutant peptides. It has been suggested that cirB is toxic to L. lactis, and that the circularin gene cluster expression under a constitutive promoter P₃₂ was not possible to achieve (Kemperman et al., 2003a). Therefore, we firstly decided to separately place the cirB gene under a nisin inducible promoter, together with pMG-CirΔB to achieve heterologous expression of circularin A in L. lactis. Later, we noticed that pMG transformants were generally less efficiently transformed in L. lactis and inclined to grow much more slowly, which would greatly impede later studies on, for example, circularin mutagenesis. To create an efficient working system for heterologous and functional expression of circularin A in L. lactis, plasmids with a nisin-inducible promotor such as pNZ8048c and pTLR4 were selected and used for gene constructions. After multiple attempts, the whole cirABCDE gene cluster was successfully inserted into a single plasmid pTLR4 via E. coli DH 5α as an intermediate host. Meanwhile, another constructed strain with the complementary design of pNZ-CirA and pTLR4-CirΔA was also achieved. Overall, L. lactis NZ9000 strains with different constructs of the circularin A gene cluster, such as pT-Cir and its variant form Cir-A +ΔA, as well as Cir-B +ΔB, were made ready for further experiments.

To assess the functionality of our different expression constructs, we performed antimicrobial assays under both non-induction and nisin-induction conditions (Figures 2B,C). Without nisin-induction, the only one that showed antimicrobial activity was the host strain with the bacteriocin gene cirA placed behind a constitutive promoter, indicating undetectable leakage of the nisin promoter in the other constructs. When induced with nisin, all three constructed strains containing the whole cirABCDE gene cluster displayed antimicrobial activity, except the control strain which lacks the structural gene cirA. Notably, the strain with cirB separately situated behind the P_nis promoter in pNZ8048c had a growth issue with a clearly altered strain morphology from other strains under the nisin-induction condition, which is likely due to the toxicity of CirB overproduction.

As shown by the antimicrobial tests, the production of the antimicrobial compound was achieved with various expression systems in L. lactis. For further confirmation, peptide expression and purification were carried out to assess the production yield and the modification level. Instead of complicated sequential steps of previously described purification methods that were commonly chosen for purification of circular bacteriocins, we applied a single-step purification by using a C18 open column. The obtained peptides from different eluent fractions were collected and subjected to an sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and MALDI-TOF analyses. The protein gel revealed a clear band from the 80% solvent elution fraction with a size close to 5K Dalton (Da) for all three expression constructs (Figure 3A). To determine the molecular size of this band, MALDI-TOF was performed and results showed a predominant peak with a detected mass of 6770.99 Da (Figure 3B). The theoretical mass of mature circularin A is 6771.05 Da, which is calculated based on the leader cleaved-off from the precursor peptide and the subsequent head-to-tail ligation. These results suggest that circularin A is fully modified in our heterologous production systems in L. lactis and that mature circularin A is eluted in fractions containing a high concentration of organic solvent (80%), which is consistent with its high hydrophobicity (GRAVY index of 1.007). Moreover, purification by TCA precipitation was also performed, and the corresponding mass of mature circularin A could be detected by MALDI-TOF, but with lower detection signal (data not shown).

Even though we successfully purified circularin A from all three expression systems in L. lactis, the production level was relatively low with our initial expression and purification conditions. To facilitate future mutagenesis studies that may significantly reduce peptide yields, we optimized the expression conditions to improve the antimicrobial production. The optimization for circularin A production mainly focused on the modifications of the growth medium, and the effect of each examined parameter was investigated independently. Compared with our standard condition, supplementation of the growth medium with additional amounts of sugar (e.g., 50 mM sucrose or 1.7% glucose) significantly increased the peptide production (Table 2 and Supplementary Figure 1). Moreover, nisin-induction from the start-point of the expression incubation also improved the peptide yield dramatically. Erythromycin, which was added to prevent the host from losing the target plasmid, had very little effect on the peptide production, and absence of glycerol in the growth medium decreased the peptide yield slightly. As expected, the control strain (ΔcirA) had no detectable antimicrobial peptide production. Based on the above observations, 2.2% glucose and immediate nisin-induction were applied to the expression conditions for subsequent peptide purification in this study. The production improvement of our two-plasmid constructed strain (Cir-A +ΔA) after optimization is shown in Supplementary Figure 2.