An Engineered Yeast Efficiently Secreting Penicillin
Figure 5
Production of β-lactam antibiotics in H. polymorpha.
(A) In vivo production of IPN by HpPen3 cells. HpPen3 cells were grown in a methanol-limited chemostat in the presence of 8 mM AAA. Cell extracts (corresponding to 1250 µl culture) were loaded in a well of a bioassay plate, which had been overlayed with the indicator strain M. luteus. After incubation of the plate, a halo, representing a zone of growth inhibition, was observed indicating that an antibiotic compound was produced. A halo was not observed when an extract of identically grown cells of the HpPen2 control strain were used. (B) In vitro production of IPN using HpPen3 cell extracts. Cell extracts were prepared from methanol grown HpPen3 cells and a volume corresponding to 500 microgram of protein was used for in vitro IPN synthesis. As a control, a desalted cell extract of P. chrysogenum DS17690 cells (250 µg protein) was used. Extracts were incubated in the presence of ATP and the amino acids AAA, L-cysteine, L-valine and subsequently loaded in a well of a bioassay plate, which had been overlayed with the indicator strain M. luteus. After incubation of the plate, a halo was observed indicating that an antibiotic compound was produced. This halo was absent in the control experiments performed without ATP or when β-lactamase was added to the reaction mixture. (C) Secretion of antibiotic compounds by HpPen4 cells. HpPen3 and HpPen4 cells were grown in continuous cultures on a mixture of glucose and methanol in the presence of 1 mM AAA and 1 mM PAA. A small aliquot (12.5 µl) of the spent medium of HpPen4, but not HpPen3 cultures, resulted in growth inhibition of the indicator strain (panels marked extracellular) on bioassay plates. Using crude cell extracts (panels marked intracellular) of HpPen3 and HpPen4 cells antibiotic compounds were detected as well. The amount of crude extracts used for the bioassay corresponded to 1250 µl of the culture volume. (D) HpPen4 cells secrete comparable amounts of antibiotics relative to P. chrysogenum NRRL1951. HpPen4 cells were grown in batch cultures on methanol in the presence of 1 mM PAA and 1 mM AAA. P. chrysogenum NRRL1951 cells were grown in batch cultures on production medium in the presence of 3 mM PAA. The figure shows that similar amounts of antibiotics are secreted by both organisms. 25 µl spent medium of both cultures was used.