Figure 1.
Schematic representation of the penicillin G (PenG) biosynthetic pathway in P. chrysogenum.
In the cytosol, the enzyme ACVS (ACVS-OH) is activated by a PPTase into ACVS-SH. The active enzyme produces ACV from the three precursor molecules AAA, L-cysteine and L-valine. IPNS subsequently converts ACV into the β-lactam IPN, which is transported into peroxisomes. In this organelle PAA is activated by PCL into phenylacetyl CoA (PA-CoA), which is used by IAT to synthesize PenG from IPN. PenG is exported from the organelle and ultimately secreted into the medium. The precursors and intermediates of PenG biosynthesis pathway are boxed.
Figure 2.
Subcellular localization of ACVS in H. polymorpha.
Immunocytochemistry using anti-ACVS antibodies showing the presence of ACVS protein in the cytosol of strain HpPen4. Cells were fixed in 3% glutaraldehyde for 1 h on ice, dehydrated in an ethanol series and embedded in Lowicryl, polymerized by UV light. Post-staining was with 0.5% uranylacetate. M – mitochondrion; P – peroxisome; V – vacuole. The bar represents 0.5 µm.
Figure 3.
Expression of genes involved in penicillin G production in H. polymorpha.
Western blot analysis demonstrating the presence of ACVS, Sfp, IPNS, IAT and PCL in cells of the various indicated H. polymorpha strains grown in batch cultures on methanol. The blots were decorated with the indicated antibodies except for Sfp, which was produced as a His6 tagged protein and detected by anti-His6 antibodies. Per lane 20 µg of protein was loaded, except for IPNS for which 2 µg of protein was used.
Figure 4.
Multiple reaction monitoring (MRM) chromatograms from ACV in extracts of strains HpPen1 and HpPen2 cells grown in batch cultures on methanol in the presence (+AAA) or absence (-AAA) of 8 mM AAA. The data show that ACV is only present in HpPen2 cells grown in the presence of AAA (lowest panel). An MRM chromatogram from the co-eluting 13C labeled ACV internal standard is included as control (upper panel).
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.
Figure 6.
H. polymorpha cells produce and secrete PenG: LC MS/MS demonstration of PEN production.
Strain HpPen4 was grown in a glucose/methanol-limited continuous culture in the presence of 1 mM AAA and 1 mM PAA. Spent medium was analyzed by LC-MS/MS. The LC chromatograms show the presence of IPN (3.45 min; Fig. 6A) and PenG (11.76 min) and a PenG-related product (7.83 min) (Fig. 6C). Fig. 6B and D show the LC-MS/MS fragmentation patterns corresponding to IPN (Fig. 6B) and PenG (Fig. 6D). The characteristic MS/MS fragmentation pattern of pure IPN and PenG and their relative abundance are represented in the tables present in Fig 6A and C.
Figure 7.
Quantification of produced β-lactam compounds in HpPen3 and HpPen4 cultures.
Strains HpPen3 and HpPen4 were grown in glucose/methanol-limited chemostat cultures in the presence of 1 mM AAA and 1 mM PAA. β-lactam compounds were quantified by IP-LC–ESI-ID-MS/MS in spent medium and cell extracts. PenG could not be detected in HpPen3 cultures, but was clearly detectable in HpPen4 cultures, where it was efficiently excreted into the medium. The PenG precursor IPN was present in both HpPen3 and HpPen4 cells; however, the amount was significantly lower in the PenG producing HpPen4 cells. Concentrations are expressed as µmol/l culture. Samples were taken in triplicate. The bars represent the standard error (SE).
Figure 8.
Deletion of PEX3 results in reduced PEN secretion.
HpPen4 and Δpex3 HpPen4 cells were grown in a glucose-limited chemostat culture supplemented with choline as nitrogen source in the presence of 1 mM PAA and 1 mM AAA. Using spent medium of Δpex3 HpPen4 cultures a smaller halo was produced relative to HpPen4. 6 µl of spent medium of both cultures was used.
Table 1.
H. polymorpha strains used in this study.
Table 2.
Plasmids used in this study.