Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host–vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine β-lyase. With this new host–vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein–PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL–SKL produced in H. polymorpha displays normal enzymatic activities.

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

<p>Abstract</p> <p>Background</p> <p>β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus <it>Penicillium chrysogenum</it>. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast <it>Hansenula polymorpha</it>. 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 <it>H. polymorpha</it>.</p> <p>Results</p> <p>The <it>P. chrysogenum pcbC </it>gene encoding isopenicillin N synthase (IPNS) was successfully expressed in <it>H. polymorpha</it>, 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 <it>P. chrysogenum</it>, in <it>H. polymorpha </it>IPNS is located in the cytosol.</p> <p>Conclusion</p> <p>In <it>P. chrysogenum</it>, 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 <it>P. chrysogenum</it>. This is a new step forward in the metabolic reprogramming of <it>H. polymorpha </it>to produce penicillin.</p

An Engineered Yeast Efficiently Secreting Penicillin

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this β-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) β-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's

Fungal feruloyl esterases: Functional validation of genome mining based enzyme discovery including uncharacterized subfamilies

Feruloyl esterases (FAEs) are a diverse group of enzymes that specifically catalyze the hydrolysis of ester bonds between a hydroxycinnamic (e.g. ferulic) acid and plant poly- or oligosaccharides. FAEs as auxiliary enzymes significantly assist xylanolytic and pectinolytic enzymes in gaining access to their site of action during biomass saccharification for biofuel and biochemical production. A limited number of FAEs have been functionally characterized compared to over 1000 putative fungal FAEs that were recently predicted by similarity-based genome mining, which divided phylogenetically into different subfamilies (SFs). In this study, 27 putative and six characterized FAEs from both ascomycete and basidiomycete fungi were selected and heterologously expressed in Pichia pastoris and the recombinant proteins biochemically characterized to validate the previous genome mining and phylogenetical grouping and to expand the information on activity of fungal FAEs. As a result, 20 enzymes were shown to possess FAE activity, being active towards pNP-ferulate and/or methyl hydroxycinnamate substrates, and covering 11 subfamilies. Most of the new FAEs showed activities comparable to those of previously characterized fungal FAEs.Peer reviewe

Fungal glucuronoyl esterases : Genome mining based enzyme discovery and biochemical characterization

4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl d-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.4-O-Methyl-D-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and therefore may play a significant role as auxiliary enzymes in biomass saccharification for the production of biofuels and biochemicals. GEs belong to a relatively new family of carbohydrate esterases (CE15) in the CAZy database (www.cazy.org), and so far around ten fungal GEs have been characterized. To explore additional GE enzymes, we used a genome mining strategy. BLAST analysis with characterized GEs against approximately 250 publicly accessible fungal genomes identified more than 150 putative fungal GEs, which were classified into eight phylogenetic sub-groups. To validate the genome mining strategy, 21 selected GEs from both ascomycete and basidiomycete fungi were heterologously produced in Pichia pastoris. Of these enzymes, 18 were active against benzyl D-glucuronate demonstrating the suitability of our genome mining strategy for enzyme discovery.Peer reviewe

Towards a penicilin producing yeast

The research included in this thesis is aimed at further expanding the knowledge of using the yeast Hansenula plymorpha as alternative cell factory for the production of ß-lactam antibiotics. The rational behing this approach is the fact that yeast species have superior fermentation characteristics over filamentous fungi. Also, if successful, this approach will provide new opportunities for highly sustainable processes using cheap substrates and the development of generic strategies to produce modified ß-lactams and/or peptide antibiotics, using the highly advanced molecular and genetic toolbox that is available for H. polymorpha. Initial studies revealed that introducing the P. chrysogenum penicillin biosynthetic pathway in yeast is paralleled by various fundamental problems of activation of these enzymes in the yeast host. Therefore we decided to follow a step by step strategy, introducing each component of the penicillin biosynthetic machinery individually in H. polymorpha and study their properties.

Presence of protein production enhancers results in significantly higher methanol-induced protein production in Pichia pastoris

Abstract Background The yeast Komagataella phaffii, better known as Pichia pastoris, is a commonly used host for recombinant protein production. Here expression vectors are reported that address the different steps of the transcription–translation–secretion pathway of heterologous protein production. Results Transcription and translation enhancing elements were introduced in an expression cassette for the production of recombinant Aspergillus niger feruloyl esterase A. The yield was increased by threefold as compared to the yield without these elements. Multiple copy strains were selected using a zeocin resistance marker in the expression cassette and showed another sixfold higher yield. Modification of the C-terminal amino acid sequence of the secretion signal did not significantly improve the production yield. Similar data were obtained for the production of another protein, recombinant human interleukin 8. Upscaling to fed-batch fermentation conditions resulted in a twofold increase for reference strains, while for strains with enhancing elements a tenfold improvement was observed. Conclusions Pichia pastoris is used for recombinant protein production in industrial fermentations. By addressing the transcription and translation of mRNA coding for recombinant protein, significant yield improvement was obtained. The yield improvement obtained under microscale conditions was maintained under fed-batch fermentation conditions. These data demonstrate the potential of these expression vectors for large scale application as improved production of proteins has major implications on the economics and sustainability of biocatalyst dependent production processes e.g. for the production of pharmaceuticals and for the bioconversions of complex molecules