Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: A Sharp Increase of the Protein Level Induces the Proliferation of Numerous,Small Protein-import Competent Peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P_AOX.PEX3^7x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm⁻³) after sucrose gradient centrifugation of WT::P_AOX.PEX3^7x homogenates, compared to normal peroxisomes (1·23 g cm⁻³). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P_AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.     

Excessive membrane development following exposure of the methylotrophic yeast Hansenula polymorpha to oleic acid-containing media

We studied the physiological responses of Hansenula polymorpha during adaptation of cells to oleic acid-containing media. Growth experiments indicated that the organism was unable to use oleic acid as the sole source of carbon and energy. However, upon incubation of glucose-grown cells in mineral media containing oleic acid, activities of various enzymes of the β-oxidation pathway were induced. These enzymes were localized in microbodies together with alcohol oxidase. Furthermore, a drastic increase in phospholipid content of the cells was observed; this was due to a rapid proliferation of membranes. These consisted of a variable number of membranous layers which were continuous with the peroxisomal membrane. Upon continued incubation, the membrane proliferations extended and large compartments were formed. This process was dependent on the presence of peroxisomes in the cells since it was not observed in peroxisome-deficient mutant strains of H. polymorpha. The newly formed membranous compartments differed from peroxisomes since they did not contain peroxisomal matrix proteins; these were confined to the single enlarged organelle which was incorporated in the membranous structure and characterized by a large alcohol oxidase crystalloid. The membranous compartments are considered to be whole entities since they could not be separated from the peroxisomes by common cell fraction methods; also they were degraded entirely after a shift of cells to glucose-excess condition. Freeze fracturing reveled that the substructure of the membranes greatly resembled that of normal peroxisomal membranes. Since a distinct enhancement of different peroxisomal membrane proteins was observed during the initial hours after the shift, we assume that exposure of H. polymorpha to acid lead to a drastic overproduction of peroxisomal membranes.     

Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: High Steady-state Levels of the Protein fully Abolish Matrix Protein Import

PEX3 encodes a 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and Δpex3 strains expressing additional copies of PEX3 under control of a substrate-inducible promoter, namely the strong alcohol oxidase (P_AOX) or the weaker amine oxidase (P_AMO) promoter. In glucose-grown Δpex3 cells, containing P_AOX. PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7–10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed. © 1997 John Wiley & Sons, Ltd.     

Assembly of amine oxidase and D-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D-alanine as the sole nitrogen source

We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D -alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D -alanine-grown cells D -amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates. The molecular masses of both amine oxidase and D -amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.     

双亲灭活米曲霉原生质体融合中原生质体制备的研究

对双亲灭活米曲霉原生质体融合育种中原生质体制备的条件进行了研究,将菌丝的预处理与细胞壁的酶解合为一步,并讨论了DTT的加入量。结果表明,原生质体制备的最佳破壁酶为纤维素酶、溶壁酶、蜗牛酶3种酶混合浓度比为5:3:1;菌丝培养15h;酶解时加入3mol/L DTT;酶解2.5h;0.8mol/L NaCl作为渗压稳定剂。所得双亲菌株原生质体的融合率为3.31%。     

Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (Δper1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of Δper1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and Δper1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in Δper1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.     

Assembly of alcohol oxidase in the cytosol of a peroxisome-deficient mutant of Hansenula polymorpha—properties of the protein and architecture of the crystals

We have studied the expression of alcohol oxidase (AO) in a peroxisome-deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose-limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild-type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3-dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol-grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha

The chimeric ChiΔH‐L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH‐L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH‐L2 yields in the peroxisome vs the cytosol. The ChiΔH‐L2 gene was yeast‐optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH‐L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH‐L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH‐L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH‐L2 production in both P. pastoris and H. polymorpha. Copyright © 2012 John Wiley & Sons, Ltd.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

微生物原生质体融合育种技术及其在发酵食品生产中的应用

微生物原生质体融合育种技术不仅为跨界融合理论提供了现实依据, 而且为实际生产提供了理论依据, 在理论研究和实际生产中都具有重要意义。微生物原生质体融合育种技术因其具有克服远缘杂交、提升优良性状和应用范围广等优点, 近年来, 在发酵食品生产中的作用日益凸显并取得了显著的成效。本文主要综述了细菌、放线菌、霉菌、酵母菌和食用菌原生质体的制备及再生, 原生质体融合方法, 原生质体融合子筛选方法以及酵母、乳酸菌和曲霉原生质体融合育种技术在发酵食品生产中的应用, 并对该技术的前景进行了探讨, 旨在为开展食品微生物优良菌株选育研究提供参考。     

Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review

Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.     

Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL-containing C-terminal sequence for targeting

Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus. In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence. For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H. polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild-type gene. Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C-terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity. From this, it was concluded that the SRL-containing C-terminus is not essential for peroxisomal targeting of AMO in H. polymorpha.     

Transient responses of Candida utilis to oxygen limitation: Regulation of the Kluyver effect for maltose

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.     

Peroxisome-deficient mutants of Hansenula polymorpha

As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.     

Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis

Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase. After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about −150mV, inside alkaline and negative. In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6·2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system. This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells. The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate. Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (ΔpH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid. The data support that the ΔpH is the main component of the proton-motive force involved in the transport of the acid and its accumulation. The lactate-proton symport stoichiometry was 1:1, being independent of the pH. Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate. However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid. These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C. utilis is inducible.     

电融合原生质体构建麦角甾醇酵母菌株的研究

实验采用细胞电融合技术进行麦角甾醇酵母菌株构建研究,在不同种属的酵母菌种进行筛选研究,确定麦角甾醇含量较高酿酒酵母(Saccharomyces cerevisiae)和细胞生物量较高产朊假丝酵母(Candi-dautilis)为亲本菌株,进行原生质体制备、原生质体再生、原生质体灭活和原生质体融合研究,确定电融合过程中电压、脉冲强度、脉冲间隙及脉冲次数等技术参数,并筛选出融合子PF-4。经发酵性能实验表明,PF-4的细胞生物量为33.17g/L,麦角甾醇含量为2.01%,均高于亲本菌株,为玉米发酵法制备麦角甾醇酵母提供生产菌株。     

细菌的原生质体融合

本文评论了有关细菌原生质体融合研究的进展,并报道了作者的部分研完工作。本文较充分讨论了原生质体融合的基本枝术步骤,包括出发株的选择,原生质体的制备,融合,再生,筛选和特性验证。介绍并推荐以育种为目的的该技术的较适使用条件。     

长枝木霉原生质体制备及再生条件研究

研究优化了长枝木霉（Trichoderma longibrachiatum）JK-15菌株菌丝体制备原生质体细胞及再生的条件。通过单因素试验和正交试验对影响原生质体形成和再生的菌龄、破壁酶组成、酶解反应温度及时间、渗透压稳定剂等因素进行了筛选优化。结果表明,长枝木霉菌株JK-15的最适条件为菌丝体菌龄18 h,混合酶组成配比为1.5%蜗牛酶∶1.5%纤维素酶∶0.1%溶壁酶,酶解条件为30 ℃条件下酶解2.5 h,原生质体制备过程中渗透压稳定剂为0.7 mol/L山梨醇,原生质体再生培养基中渗透压稳定剂为0.7 mol/L蔗糖,实验结果为长枝木霉JK-15的育种工作奠定了良好的技术基础。