Genetically Engineered Yeast for Enhanced Biodegradation of Β-lactam Antibiotics

Abstract

High environmental concentrations of pharmaceuticals, particularly antibiotics, have been observed worldwide, while antibiotic concentrations in the ng/L to µg/L range may adversely affect biota and contribute to the formation of antibiotic resistance. The underlying causes of high environmental concentrations include, in addition to the low rate of antibiotic' metabolization and the high rate of usage, especially of β-lactam antibiotics, the insufficient removal by conventional wastewater treatment methods. Consequently, alternative methods need to be developed to remove antibiotics from wastewater-one possibility is the use of enzymes. In this study, the enzyme β-lactamase was secreted by a genetically modified yeast (Saccharomyces cerevisiae) upon recognition of a pheromone (α-factor) as inducer to enable the degradation of ampicillin. This represents a crucial step on the road to a sensor-actuator system, allowing for the development of an intelligent removal system that can react to the presence of antibiotics. Ampicillin and its transformation products were studied by LC-MS/MS measurements using a carbamoyl functionalized column under hydrophilic interaction liquid chromatography (HILIC) conditions, which allowed detection of ampicillin at concentrations of 2.43 nM. The dependence of ampicillin degradation on α-factor concentration and the cultivation time of the yeast was demonstrated, resulting in higher degradation rate with higher α-factor concentrations and longer yeast cultivation times. Over 90% of 10 µM ampicillin was degraded within 0.5 h using 250 nM α-factor and a cultivation period of 24 h. Finally, the transferability to other β-lactam antibiotics was investigated, resulting in complete degradation of amoxicillin, penicillin G and piperacillin within 24 h.

Keywords: Antibiotics; Enzymatic degradation; LC–MS/MS; β-lactamase.

Fig. 1

Scheme of the yeast plasmid p426-FIG1-HSP150(SS)-TEM1. The β-lactamase coding sequence (TEM1) was fused to a secretion signal (HSP150(SS)) and set under the control of the α-factor dependent FIG1 promotor. The construct was integrated into the high-copy vector p426 [37] to facilitate a high-level production and secretion of β-lactamase upon addition of α-factor

Fig. 2

Expression analyses with p426FIG1-HSP150(SS)-TEM1. Yeast was cultivated in W0 medium in the presence of 250 nM α-factor (+ α) or absence (-α) and samples were taken at different time points after induction. Cell extracts (a) as well as culture supernatants (b) were separated by 12% SDS-PAGE and Western Blot analysis was performed using antibodies against TEM1p (upper panels). Total protein (20 µg per lane) was visualized using colloidal Coomassie staining (lower panels)

Fig. 3

Influence of cultivation period on the presence of β-lactamase in the supernatant. Detection of β-lactamase content was performed indirectly through the degradation of 10 µM AMP—analyzed by (a) LC–MS/MS and (b) measurement of absorbance at 486 nm in the Nitrocefin assay. S. cerevisiae was cultivated in W0-medium. Data represent mean values ± standard deviation from three independent experiments

Fig. 4

Influence of different α-factor concentrations on the secretion of β-lactamase in the supernatant of S. cerevisiae cultures in W0 medium following 4 h of cultivation. Expression of TEM1 in yeast was induced using the α-factor concentrations indicated in the inset. Concentration of AMP was measured (a) indirectly by analysing the degradation of 10 µM AMP over a 24-h period in the 1:50 diluted supernatant. The values obtained from three different yeast cultivations and are normalized to c_0. (b) Further analyses of the same supernatant were carried out using the nitrocefin assay. The supernatant was diluted 1:5 before starting the experiment. The blank (media) was subtracted. Data represent mean values ± standard deviation (n = 3)

Fig. 5

Formation of transformation products from AMP (a) Pathway for the generation of transformation products m/z = 368 and m/z = 324 from AMP—cleavage sites marked and (b) TP formation, measured in the form of detected peak areas for AMP, TP368 and TP324; analysis conducted over a degradation period of 24 h. S. cerevisiae was cultivated for 24 h in W0 medium. The supernatant was harvested, diluted 1:50 and then used in the degradation study for TP analysis. AMP and the transformation products m/z = 368 and m/z = 324 were detected

Fig. 6

Degradability of different antibiotics using supernatant containing TEM1p. Prior to beginning the timecourse, S. cerevisiae was cultivated in W0 medium containing 250 nM α-factor for 24 h. The supernatant obtained after 24 h was diluted 1:50, and 10 µM AMP, AMX, CLX, CEL, PEN G or PIP (in separate vials) were added to analyze degradability over a period of 24 h. Data represent mean values ± standard deviation for the three independent experiments. Furthermore, structural formulae of Ampicillin (AMP), Amoxicillin (AMX), Penicillin G (PEN G), Piperacillin (PIP), Cloxacillin (CLX) and Cefalotin (CET) is shown. The β-lactam ring is highlighted in yellow