Bioengineering the Antimicrobial Activity of Yeast by Recombinant Thanatin Production

Abstract

The global spread of antibiotic resistance marks the end of the era of conventional antibiotics. Mankind desires new molecular tools to fight pathogenic bacteria. In this regard, the development of new antimicrobials based on antimicrobial peptides (AMPs) is again of particular interest. AMPs have various mechanisms of action on bacterial cells. Moreover, AMPs have been reported to be efficient in preclinical studies, demonstrating a low level of resistance formation. Thanatin is a small, beta-hairpin antimicrobial peptide with a bacterial-specific mode of action, predetermining its low cytotoxicity toward eukaryotic cells. This makes thanatin an exceptional candidate for new antibiotic development. Here, a microorganism was bioengineered to produce an antimicrobial agent, providing novel opportunities in antibiotic research through the directed creation of biocontrol agents. The constitutive heterologous production of recombinant thanatin (rThan) in the yeast Pichia pastoris endows the latter with antibacterial properties. Optimized expression and purification conditions enable a high production level, yielding up to 20 mg/L of rThan from the culture medium. rThan shows a wide spectrum of activity against pathogenic bacteria, similarly to its chemically synthesized analogue. The designed approach provides new avenues for AMP engineering and creating live biocontrol agents to fight antibiotic resistance.

Keywords: antibiotic resistance; antimicrobial peptides; recombinant antibiotics; thanatin; yeast biocontrol agents.

Figure 1

(A) A schematic representation of the rThan expression vector. P_GAP—GAP promoter, aMF—secretion signal sequence, rThan—thanatin coding sequence, 3′AOX TT—AOX1 transcriptional terminator, HIS4—histidinol dehydrogenase, KanR—yeast kanamycin resistance, AmpR—E. coli ampicillin resistance, Ori—E. coli origin of replication. (B) A schematic representation of the yeast bioengineering workflow. Yeast cells were transformed with the target plasmid. The resulting colonies were overlaid with bacteria-inoculated agar. Clones with clear growth inhibition zones were used for further investigations. The control plasmid containing fluorescent protein mCherry was described previously [35]. Agar overlay was performed with E. coli ΔtolC as the target bacteria. (C) Antimicrobial activity of rThan-producing yeasts, estimated by agar overlay assay using target strains E. coli ΔlptD, E. coli ΔtolC, and E. coli BL21(DE3). The rThan-producing yeasts formed transparent zones of inhibition.

Figure 2

Production and purification analysis of rThan: (A)—Time-dependent rThan production analysis, where columns represent rThan concentrations in culture media at different time points; (B)—Tricine-SDS-PAGE analysis of rThan purification: MW—Protein MW marker; FT—SP-sepharose flowthrough; 400—concentration of NaCl in elution buffer(mM); 500–650—part of a linear gradient of an elution buffer with increasing NaCl concentration from 500 to 650 mM. Data represent the mean of three biological replicates ± SD.

Figure 3

Chemical synthesis of thanatin. (A) HPLC profile of crude linear thanatin (53% purity): C18 column Phenomenex Luna (130 Å, 3.5 μm, 4.6 × 250 mm); temperature: 35 °C; flow: 1.0 mL/min; eluent: 0.1% (v/v) TFA in H₂O (buffer A) and 0.1% (v/v) TFA in CH₃CN (buffer B), λ 220 nm; gradient: 5−60% of buffer B in 20 min. (B) HPLC profile of purified cyclized thanatin (95.7% purity): C18 column Phenomenex Luna (130 Å, 3.5 μm, 4.6 × 250 mm); temperature: 35 °C; flow: 1.0 mL/min; eluent: 0.1% (v/v) TFA in H₂O (buffer A) and 0.1% (v/v) TFA in CH₃CN (buffer B), λ 220 nm; gradient: 5−60% buffer B in 20 min. (C) MALDI-TOF mass spectrum of cyclized sThan: m/z 2433.34—experimental data for [M + H]⁺, 2433.28—theoretical.

Figure 4

Minimal inhibitory concentrations (MICs) of rThan and sThan toward a panel of Gram-negative pathogens and model Escherichia coli strains.

Figure 5

E. coli ΔtolC sfGFP growth inhibition landscape. rThan-producing P. pastoris was cocultivated with GFP-producing E. coli ΔtolC at different starting cell concentrations. Growth inhibition of target bacteria was estimated according to the level of the GFP fluorescence signal: from complete growth inhibition (black color) to no inhibition (light yellow color). CFU/mL data indicate initial cell concentrations. The detection limit of viable E. coli ΔtolC sfGFP cells is approximately 10³ CFU/mL.