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.

A Peroxisomal Lon Protease and Peroxisome Degradation by Autophagy Play Key Roles in Vitality of Hansenula polymorpha Cells

In eukaryote cells various mechanisms exist that are responsible for the removal of non-functional proteins. Here we show that in the yeast Hansenula polymorpha (H. polymorpha) a peroxisomal Lon protease, Pln, plays a role in degradation of unfolded and non-assembled peroxisomal matrix proteins. In addition, we demonstrate that whole peroxisomes are constitutively degraded by autophagy during normal vegetative growth of WT cells. Deletion of both H. polymorpha PLN and ATG1, required for autophagy, resulted in a significant increase in peroxisome numbers, paralleled by a decrease in cell viability relative to WT cells. Also, in these cells and in cells of PLN and ATG1 single deletion strains, the intracellular levels of reactive oxygen species had increased relative to WT controls. The enhanced generation of reactive oxygen species may be related to an uneven distribution of peroxisomal catalase activities in the mutant cells, as demonstrated by cytochemistry. We speculate that in the absence of HpPln or autophagy unfolded and non-assembled peroxisomal matrix proteins accumulate, which can form aggregates and lead to an imbalance in hydrogen peroxide production and degradation in some of the organelles.

ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes

ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also an analysis of Arabidopsis thaliana and Homo sapiens databases was included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. The A. thaliana and human genomes encode multiple proteins highly similar to specific fungal Atg proteins (paralogs), possibly representing cell type-specific isoforms. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, Atg19, the receptor of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha pex14 null mutant

Hansenula polymorpha Delta pex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol, Here, we show that in Delta pex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Delta pex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Delta pex14 cells overproducing Pex5p, Pex5P had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p

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

Boundary layer convective-like activity at Dome Concordia, Antarctica

The paper presents the micro-meteorological field experiment carried out at the plateau station of Dome Concordia (3300 m a.s.l.) during the Antarctic summer of 1997. The experiment dealt with the study of the trends of boundary layer features and the characteristics of the surface energy and momentum exchanges. A monostatic Doppler sodar, fast-response sensors and radiometers were used for this study. The experiment was part of a program that aims to assess the role of the continental polar regions in shaping the surface circulation over Antarctica. In spite of the markedly stable conditions found throughout the investigated period, some convective-like activity was detected during the warmer hours of the day

Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha

AbstractATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete

Obstruction of polyubiquitination affects PTS1 peroxisomal matrix protein import

AbstractPex4p is an ubiquitin-conjugating enzyme that functions at a late stage of peroxisomal matrix protein import. Here we show that in the methylotrophic yeast Hansenula polymorpha production of a mutant form of ubiquitin (UbK48R) has a dramatic effect on PTS1 matrix protein import. This effect was not observed in cells lacking Pex4p, in which the peroxisome biogenesis defect was largely suppressed. These findings provide the first indication that the function of Pex4p in matrix protein import involves polyubiquitination. We also demonstrate that the production of UbK48R in H. polymorpha results in enhanced Pex5p degradation. A similar observation was made in cells in which the PEX4 gene was deleted. We demonstrate that in both strains Pex5p degradation was due to ubiquitination and subsequent degradation by the proteasome. This process appeared to be dependent on a conserved lysine residue in the N-terminus of Pex5p (Lys21) and was prevented in a Pex5pK21R mutant. We speculate that the degradation of Pex5p by the proteasome is important to remove receptor molecules that are stuck at a late stage of the Pex5p-mediated protein import pathway

High-intensity interval training in polycystic ovary syndrome : A two-center, three-armed randomized

Purpose Exercise training is recommended to improve cardiometabolic health and fertility in women with polycystic ovary syndrome (PCOS), yet there are few randomized controlled trials on the effects of different exercise protocols on clinical reproductive outcomes. Our aim was to determine the effect of high-intensity interval training (HIT) on menstrual frequency, as a proxy of reproductive function, in women with PCOS. Methods The IMPROV-IT study was a two-center randomized controlled trial undertaken in Norway and Australia. Women with PCOS were eligible for inclusion. After stratification for body mass index <27 or ≥27 kg·m−2 and study center, participants were randomly allocated (1:1:1) to high-volume HIT (HV-HIT), low-volume HIT (LV-HIT), or a control group. Measurements were assessed at baseline, after the 16-wk exercise intervention, and at 12-month follow-up. The primary outcome was menstrual frequency after 12 months. Secondary outcomes included markers of cardiometabolic and reproductive health, quality of life, and adherence to and enjoyment of HIT. Results We randomly allocated 64 participants to the HV-HIT (n = 20), LV-HIT (n = 21), or control group (n = 23). There were no differences in menstrual frequency at 12 months between the LV-HIT and control groups (frequency ratio, 1.02; 95% confidence interval [CI], 0.73–1.42), the HV-HIT and control groups (frequency ratio, 0.93; 95% CI, 0.67–1.29), or the LV-HIT and HV-HIT groups (frequency ratio, 1.09; 95% CI, 0.77–1.56). Menstrual frequency increased in all groups from baseline to 12 months. More participants became pregnant in the LV-HIT group (n = 5) than in the control group (n = 0, P = 0.02). Conclusions A semisupervised HIT intervention did not increase menstrual frequency in women with PCOS. Clinical Trial Registration Number:ClinicalTrials.gov (NCT02419482)