Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha
© Gidijala et al; licensee BioMed Central Ltd. 2008
Received: 25 October 2007
Accepted: 19 March 2008
Published: 19 March 2008
β-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.
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°C). Heterologously produced IPNS protein levels were enhanced when the cultivation temperature was lowered to either 25°C or 30°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.
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°C, the optimal growth temperature for P. chrysogenum. This is a new step forward in the metabolic reprogramming of H. polymorpha to produce penicillin.
β-lactam antibiotics like penicillins and cephalosporins belong to one of the largest-selling classes of drugs worldwide with a production of forty-five thousand tons in the year 2000 . Penicillins and cephalosporins are produced by the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum, respectively, as well as some filamentous bacteria. These antibiotics possess as common structural motif the β-lactam ring . Not surprisingly, the penicillin and cephalosporin biosynthetic pathways have the first two enzymatic steps in common. First, the non-ribosomal peptide synthetase δ(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) forms the tripeptide ACV. The formation of ACV acts as the committed step in both penicillin and cephalosporin biosynthesis. ACV is subsequently converted into isopenicillin N (IPN), which has the characteristic β-lactam backbone, by the enzyme isopenicillin N synthase (IPNS). Both ACVS and IPNS have been shown to be located in the cytosol in P. chrysogenum [3, 4]. Subsequent replacement of the α-amino adipoyl side chain of IPN by the more hydrophobic phenylacetyl or phenoxyacetyl moieties occurs in P. chrysogenum in the specific environment of the microbody and results in the formation of penicillin [3, 5]. On the other hand, epimerization of the α-amino adipoyl moiety followed by ring expansion leads to cephalosporin biosynthesis in A. chrysogenum . So far, a requirement for specific organelles for cephalosporin production in this filamentous fungus is unknown.
Isopenicillin N synthase (IPNS) belongs to a class of non-heme ferrous iron dependent oxidoreductases. During its enzymatic reaction one molecule of oxygen is completely transformed into two water molecules by removal of four hydrogen atoms from the ACV tripeptide . Detailed mechanistic studies of the IPNS enzyme were carried out using Aspergillus nidulans IPNS produced in Escherichia coli [8, 9]. These studies showed that the formation of the β-lactam ring is carried out by an iron (IV)-oxy intermediate with the His212, Asp214, and His268 residues of A. nidulans IPNS forming the active center. Furthermore, a conserved Arg-Xxx-Ser oxidase motif is involved in binding of the ACV substrate .
Our objective is to introduce the penicillin biosynthesis pathway from P. chrysogenum 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. Additionally, introducing the penicillin biosynthesis pathway into this yeast species allows a better understanding of the function of microbodies in penicillin production. In the past, the microbody-localized proteins involved in penicillin biosynthesis in P. chrysogenum – isopenicillin N:acyl CoA acyltransferase (IAT) and phenylacetyl-CoA ligase (PCL) – were successfully produced in H. polymorpha in an active form [11, 12]. Here we present our data on the expression of the P. chrysogenum pcbC gene encoding IPNS in H. polymorpha.
Results and Discussion
IPNS produced in H. polymorphaat 37°C is not stable
Effect of the cultivation temperature on IPNS production in H. polymorpha
Instability of heterologous produced protein in yeast can be attributed to improper folding in the unnatural host, or to absence of activation, both of which can result in turn-over of the protein . Our assumption was that P. chrysogenum IPNS was not properly folding in H. polymorpha, which might be overcome by cultivating the recombinant strain at a reduced temperature. Since P. chrysogenum hyphae are usually grown at 25°C, we cultivated H. polymorpha strain IPNS 4.2 on methanol media at 30°C and 25°C again taking aliquots at different stages of growth (Fig. 1, Panels C and E). Under these conditions, the growth rate of the H. polymorpha cells is reduced. Western blots demonstrated that IPNS protein was present in H. polymorpha extracts at all stages of growth (Fig. 1, Panels D and F). We also analyzed the effect of the reduction in the growth temperature of strain IPNS 4.2 on the stability of IPNS. Cells grown at 30°C and 25°C on methanol media to the mid-exponential growth phase were shifted to 0.5% glucose in order to repress the P AOX and aliquots of equal volume were taken at regular time intervals after the shift. Western blot analysis demonstrated that IPNS protein was fully stable when produced at the lower growth temperatures (Fig. 2, Panels B and C).
H. polymorphaIPNS 4.2 cells grown at reduced temperatures produce functionally active IPNS
Localization of IPNS produced in H. polymorpha
In P. chrysogenum IPNS is a cytosolic protein . Since two of the enzymes involved in penicillin biosynthesis (IAT and PCL) are located to microbodies in P. chrysogenum  [W.H. Meijer et al., unpublished results], it is important to know the subcellular location of IPNS in H. polymorpha. Cells of strain IPNS 4.2 were cultivated on methanol to induce IPNS production and prepared for immunocytochemistry using polyclonal α-IPNS antiserum. Immunogold labelling was only observed in the cytosol or on nuclear profiles (Fig. 5) as is characteristic for soluble proteins . Microbodies and mitochondria invariably lacked specific labelling, which led us to conclude that IPNS produced in H. polymorpha is located in the cytosol.
Previously, we succeeded in producing IAT and PCL in H. polymorpha and to target these proteins to the correct subcellular location, the microbody [11, 12]. Both proteins showed enzymatic activity in H. polymorpha cells cultured at 37°C, implying proper folding at the physiological growth temperature of the heterologous host. Nevertheless, since both proteins originate from P. chrysogenum, optimal enzyme activities are expected at 25°C. The observation that active IPNS is only produced in H. polymorpha cells cultured at 25°C implies that penicillin production in this heterologous host may only be successful at reduced growth temperatures.
Our next step on the way to insert the entire penicillin biosynthesis pathway into H. polymorpha will be the production of functionally active ACVS in the heterologous host. This undoubtedly is the most challenging step, since yeast genomes do not encode non-ribosomal peptide synthetases like ACVS, while in bacteria and filamentous fungi these proteins are widespread.
Microorganisms and growth conditions
H. polymorpha strains used are derivatives of NCYC495 ade11.1 leu1.1  and were grown at 37°C, 30°C or 25°C in either (i) rich complex media (YPD) containing 1% yeast extract, 1% peptone and 1% glucose, (ii) selective media containing 0.67% yeast nitrogen base without amino acids (DIFCO) supplemented with 0.5% glucose (YND), or (iii) mineral medium (MM) as described by Van Dijken et al. , supplemented with 0.25% ammonium sulphate using 0.5% glucose or 0.5% methanol as carbon source. For growth on plates, 2% granulated agar was added to the media. Whenever necessary, media were supplemented with 30 μg/ml leucine and 20 μg/ml adenine. For biochemical analysis, selected strains were pre-cultured at least three times in MM containing glucose and subsequently shifted to MM containing methanol to induce expression of genes under the control of the P AOX .
A high penicillin producing P. chrysogenum strain (DSM anti-infectives, Delft, The Netherlands) was used as a control and was grown for 48 hours on a defined penicillin production medium supplemented with 0.05% phenylacetic acid .
For cloning purposes, Escherichia coli DH5α (Gibco-Brl, Gaithesburg, MD) was used and grown at 37°C in LB medium (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl), supplemented with 50 μg/ml kanamycin when required.
Miscellaneous DNA techniques
All DNA manipulations were carried out according to standard methods . H. polymorpha cells were transformed by electroporation . DNA modifying enzymes were used as recommended by the supplier (Roche, Almere, The Netherlands). Pwo polymerase was used for preparative polymerase chain reactions (PCR). Oligonucleotides were synthesized by Life Technologies (Breda, The Netherlands). DNA sequencing reactions were performed at BaseClear (Leiden, The Netherlands). For DNA sequence analysis, the Clone Manager 5 program (Scientific and Educational Software, Durham, USA) was used.
Construction of H. polymorphaIPNS 4.2
For the construction of a pcbC expression plasmid a HindIII site was introduced at the 5' end and a SalI site at the 3'end of P. chrysogenum pcbC by PCR with the primers ipns-F (5' ACTAAGCTTATGGCTTCCACCCCCAAG 3') and ipns-R (5' ATCGTCGACTCATGTCTGGCCGTTCTTGTTG 3') using DNA from a P. chrysogenum cDNA library as template . The resulting 1002 bp DNA fragment was digested with HindIII and SalI and cloned between the HindIII and SalI sites of pHIPX4  resulting in plasmid pHIPX4-pcbC. The nucleotide sequence of the cloned PCR fragment was confirmed by sequencing (data not shown). Subsequently, pHIPX4-pcbC was linearized with SphI in the P AOX region and transformed into H. polymorpha NCYC495 ade11.1 leu1.1 cells. Fast growing leucine prototrophic transformants were selected, which are expected to carry multiple copies of the expression cassette . Correct integration was confirmed by colony PCR using the genome-specific primer Aox-F (5' TCACACCGTAACGCTTTATCGCC 3'), which corresponds to a region directly upstream of the AOX promoter and the plasmid-specific primer pcbC-R (5' ATCTGGTCCTGGTGCTCCTTG 3'), which corresponds to the 5'end of the pcbC coding sequence. This will result in the formation of a product of 1898 bp exclusively in correct integrants (data not shown), one of which was designated IPNS 4.2.
Crude extracts of H. polymorpha cells were prepared with glass beads basically as described previously . Crude extracts of P. chrysogenum hyphae were prepared from 150 mg of lyophilized mycelium . For Western blots, extracts of H. polymorpha and P. chrysogenum cells were prepared using the TCA method . Protein concentrations were determined using the Bio-Rad Protein Assay system with bovine serum albumin as standard. SDS-PAGE and Western blotting were performed by established procedures. For Western blots H. polymorpha cells of the indicated strains were cultivated on methanol-containing media to induce the expression of the AOX promoter. At the indicated time intervals, aliquots of cells were taken and TCA precipitated. Extracts were prepared for Western blotting and decorated with polyclonal IPNS-specific antibodies . Equal amounts of protein were loaded per lane, unless indicated otherwise.
Bioassay for isopenicillin N synthase enzyme activity
Isopenicillin N synthase activity was determined in crude cell extracts prepared with extraction buffer (50 mM Tris-HCl, pH 8.0, 0.3 mM DTT, 1 mM PMSF) instead of phosphate buffer. Cell extracts corresponding to a fixed amount of protein (10 to 50 μg) were incubated at 25°C for 10 min in a final volume of 200 μl with 0.75 mM reduced Bis-ACV (Bachem Fein Chemicalien AG, Switzerland), 0.43 mM of FeSO4.7H2O as cofactor and 14.1 mM L-ascorbic acid as additive. After incubation, the reaction was terminated by addition of an equal volume of methanol to precipitate the proteins. After centrifugation, equal volume fractions of the supernatants were loaded in a well on an bioassay plate on which the indicator strain Micrococcus luteus ATCC 9341 had been plated. Plates were incubated overnight at 30°C. When penicillinase was used to degrade β-lactams in the reaction mixture, this was added (50,000 IU per reaction) prior to termination of the reaction with methanol.
Intact cells were prepared for immunocytochemistry as described previously . Ultrathin sections of unicryl-embedded cells were labeled using polyclonal antiserum raised in rabbit against IPNS and goat-anti-rabbit antibodies conjugated to gold according to the instruction of manufacturers (Amersham UK).
LG and JAKWK are financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations  through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability). We would like to thank Ron. S. Booij for skillful technical assistance.
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