Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha

Abstract

Background: beta-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha.

Results: The P. chrysogenum pcbC gene encoding isopenicillin N synthase (IPNS) was successfully expressed in H. polymorpha, but the protein produced was unstable and inactive when the host was grown at its optimal growth temperature (37 degrees C). Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25 degrees C or 30 degrees C. Furthermore, IPNS produced at these lower cultivation temperatures was functionally active. Localization experiments demonstrated that, like in P. chrysogenum, in H. polymorpha IPNS is located in the cytosol.

Conclusion: In P. chrysogenum, the enzymes involved in penicillin production are compartmentalized in the cytosol and in microbodies. In this study, we focus on the cytosolic enzyme IPNS. Our data show that high amounts of functionally active IPNS enzyme can be produced in the heterologous host during cultivation at 25 degrees C, the optimal growth temperature for P. chrysogenum. This is a new step forward in the metabolic reprogramming of H. polymorpha to produce penicillin.

Figure 1

Production of IPNS by H. polymorpha strain IPNS 4.2 cultivated at various temperatures. Western blots showing IPNS production in cells of the H. polymorpha strain IPNS 4.2 cultivated at 37°C (panels A and B), 30°C (panels C and D) and 25°C (panels E and F). Aliquots were taken at different stages during cultivation at approximately equal optical densities (expressed as A₆₆₀ and indicated as 1, 2, 3, and 4 in panels A, C and E). IPNS is produced in strain IPNS 4.2 at all growth temperatures. However, during later stages of growth relatively low IPNS protein levels were observed in cells grown at 37°C, while at 30°C and 25°C enhanced levels of IPNS protein were detected.

Figure 2

Stability of IPNS produced by H. polymorpha strain IPNS 4.2 cultivated at various temperatures. H. polymorpha IPNS 4.2 cells were cultivated on methanol media to the mid-exponential growth phase (A₆₆₀ of approximately 1.5) at 37°C, 30°C and 25°C (panels A, B, and C, respectively). Subsequently, 0.5% glucose was added to repress the AOX promoter and stop further IPNS synthesis. Aliquots of the cultures were taken at the indicated time intervals after the shift. Western blots were prepared using equal volume fractions loaded per lane. For clarity, in panels B and C protein samples were diluted 10 times prior to loading. The results show that in IPNS 4.2 cells cultivated at 37°C IPNS protein is unstable since little protein is detected 1 h after the shift. In cells cultivated at lower temperatures, IPNS protein remains detectable at all time points.

Figure 3

Comparison of IPNS protein produced by H. polymorpha IPNS 4.2 cultivated at various temperatures. Western blot showing the relative amounts of IPNS protein produced by mid-exponentially grown H. polymorpha IPNS 4.2 cells cultivated at 37°C, 30°C and 25°C (lanes 3, 4 and 5, respectively). As controls, H. polymorpha WT cells, cultivated on methanol at 25°C (lane 2), and P. chrysogenum cells, cultivated for 40 h on penicillin production media supplemented with phenylacetic acid (lane 1), are included. These demonstrate the specificity of the α-IPNS antiserum.

Figure 4

IPNS produced by H. polymorpha strain IPNS 4.2 cultivated at 25°C is enzymatically active. H. polymorpha IPNS 4.2 cells were cultivated on methanol-containing media to the mid-exponential growth phase at 37°C (well 4), 30°C (well 2) and 25°C (well 3). As controls, H. polymorpha WT cells, cultivated on methanol at 25°C (well 5), and P. chrysogenum cells, cultivated for 40 h on penicillin production media supplemented with phenylacetic acid (well 1) were used. Cell free extracts (40 μg of protein each) were incubated for 20 min at 25°C in a buffer containing ACV to allow production of IPN. Panel A: Reactions were terminated by methanol addition. After protein precipitation, an equal volume of reaction product was added to each well of a bioassay plate on which M. luteus had been plated as indicator strain. The results indicate that extracts prepared from IPNS 4.2 cells cultivated at 25°C and 30°C contain active IPNS enzyme, resulting in the formation of a halo. In contrast, extracts from IPNS 4.2 cells grown at 37°C show no activity. Panel B: As control, the reaction products were further incubated in the presence of penicillinase for an additional 30 min at 25°C. After that, the reaction mixtures were terminated by methanol addition and the reaction products analyzed on a bioassay plate as described in A. The absence of halos on the plate demonstrates that the IPNS 4.2 extracts indeed allow formation of the β-lactam ring.

Figure 5

Subcellular localization of IPNS protein produced in H. polymorpha. Methanol-grown H. polymorpha IPNS 4.2 cells were prepared for immunocytochemistry, using α-IPNS-specific antiserum. Gold particles are predominantly observed on the cytosol, but not on peroxisomes or mitochondria. Key: M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. The bar represents 0.5 μm.