Tools for genetic engineering of the yeast Hansenula polymorpha.

Hansenula polymorpha is a methylotrophic yeast species that has favorable properties for heterologous protein production and metabolic engineering. It provides an attractive expression platform with the capability to secrete high levels of commercially important proteins. Over the past few years many efforts have led to advances in the development of this microbial host including the generation of expression vectors containing strong constitutive or inducible promoters and a large array of dominant and auxotrophic markers. Moreover, highly efficient transformation procedures used to generate genetically stable strains are now available. Here, we describe these tools as well as the methods for genetic engineering of H. polymorpha.

Targeting carbonic anhydrase IX by nitroimidazole based sulfamides enhances the therapeutic effect of tumor irradiation: a new concept of dual targeting drugs.

BACKGROUND AND PURPOSE: Carbonic anhydrase IX (CAIX) plays an important role in pH regulation processes critical for tumor cell growth and metastasis. We hypothesize that a dual targeting bioreductive nitroimidazole based anti-CAIX sulfamide drug (DH348) will reduce tumor growth and sensitize tumors to irradiation in a CAIX dependent manner. MATERIAL AND METHODS: The effect of the dual targeting anti-CAIX (DH348) and its single targeting control drugs on extracellular acidification and radiosensitivity was examined in HT-29 colorectal carcinoma cells. Tumor growth and time to reach 4× start volume (T4×SV) was monitored for animals receiving DH348 (10 mg/kg) combined with tumor single dose irradiation (10 Gy). RESULTS: In vitro, DH348 reduced hypoxia-induced extracellular acidosis, but did not change hypoxic radiosensitivity. In vivo, DH348 monotherapy decreased tumor growth rate and sensitized tumors to radiation (enhancement ratio 1.50) without systemic toxicity only for CAIX expressing tumors. CONCLUSIONS: A newly designed nitroimidazole and sulfamide dual targeting drug reduces hypoxic extracellular acidification, slows down tumor growth at nontoxic doses and sensitizes tumors to irradiation all in a CAIX dependent manner, suggesting no "off-target" effects. Our data therefore indicate the potential utility of a dual drug approach as a new strategy for tumor-specific targeting.

Novel genetic tools for Hansenula polymorpha.

Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H. polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H. polymorpha. For this, we generated a yku80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H. polymorpha.

Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1.

We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.

Peroxisomes as dynamic organelles: peroxisome abundance in yeast.

Peroxisomes are cell organelles that are present in almost all eukaryotic cells and involved in a large range of metabolic pathways. The organelles are highly dynamic in nature: their number and enzyme content is highly variable and continuously adapts to prevailing environmental conditions. This review summarizes recent relevant developments in research on processes that are involved in the regulation of peroxisome abundance and maintenance. These processes include fission of the organelles, formation of new peroxisomes from the endoplasmic reticulum, autophagic degradation and segregation/inheritance during cell division.

A conserved function for Inp2 in peroxisome inheritance.

In budding yeast Saccharomyces cerevisiae, the peroxisomal protein Inp2 is required for inheritance of peroxisomes to the bud, by connecting the organelles to the motor protein Myo2 and the actin cytoskeleton. Recent data suggested that the function of Inp2 may not be conserved in other yeast species. Using in silico analyses we have identified a weakly conserved Inp2-related protein in 18 species of budding yeast and analyzed the role of the identified protein in the methylotrophic yeast Hansenula polymorpha in peroxisome inheritance. Our data show that H. polymorpha Inp2 locates to peroxisomes, interacts with Myo2, and is essential for peroxisome inheritance.

Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation.

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells. Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.