Effects of methanol on expression of an anticoagulant hirudin in recombinant Hansenula polymorpha

A series of batch, fed-batch, and continuous cultures was carried out to analyze the effects of methanol on the fermentation characteristics of recombinant Hansenula polymorpha for the production of hirudin, an anticoagulant. Hirudin expression efficiencies were greatly influenced by the methanol concentrations in continuous and fed-batch culture modes. At a steady state of continuous culture, an optimum methanol concentration of 1.7 g l⁻¹ was determined at a dilution rate of 0.18 h⁻¹ with 1.8 mg l⁻¹ h⁻¹ hirudin productivity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 58–61. Received 21 September 2000/ Accepted in revised form 10 June 2001     

Production of recombinant hirudin in Hansenula polymorpha: variation of gene expression level depends on methanol oxidase and fermentation strategies

Various recombinant Hansenula polymorpha strains were developed and compared for their level of expression of the anticoagulant hirudin. H. polymorpha DL1-57 harboring an autonomously replicating sequence, HARS36, efficiently expressed the gene for recombinant hirudin. The effect of methanol oxidase (MOX) on the expression of the hirudin gene in H. polymorpha DL1-57 was studied, and the fermentation strategies coupled with the MOX activity and an antioxidant, tocopherol, were also examined. Received 4 February 1998/ Accepted in revised form 24 June 1998     

High-level expression of foreign genes in Hansenula polymorpha

The methylotrophic yeast Hansenula polymorpha belongs to a limited number of non-Saccharomyces yeast species used as hosts for heterologous gene expression. It has successfully been applied for the production of hormones, antigens and enzymes. The system excells by mitotically stable recombinant strains, high productivity and faithful processing of the produced polypeptides. The favourable characteristics of this microorganism for protein production at an industrial scale are described in the following article focusing on some recent representative examples.     

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 beta-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.     

Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides

 We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (P_AOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (±78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment. Received: 16 June 1995 / Accepted: 20 September 1995     

Peroxisome homeostasis in Hansenula polymorpha

Peroxisomes are essential organelles in many eukaryotes. Until recently, the main focus of the investigations concerning these important organelles was to understand the biogenesis of the peroxisome (induction, proliferation and matrix protein import). However, when peroxisomes become redundant they are quickly degraded by highly selective processes known as pexophagy. The first molecular studies on pexophagy have indicated that this process shares many features with certain transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole targeting and endocytosis). Nevertheless, recent data demonstrate that in addition to common genes also unique genes are required for these transport processes. The main focus for the future should therefore be on identifying the unique determinants of pexophagy. Earlier results suggest that in the methylotrophic yeast Hansenula polymorpha proteins located on the peroxisome itself are required for pexophagy. Thus, it has become essential to study in detail the role of peroxisomal membrane proteins in the degradation process. This review highlights the main achievements of the last few years, with emphasis on H. polymorpha.     

多形汉逊酵母研究进展

作为研究甲醇代谢、过氧化物酶体稳态和硝酸盐吸收的模式生物,多形汉逊酵母近年来在基础研究领域日益受到重视。在工程应用领域,利用多形汉逊酵母表达真核外源基因有特殊的优势。譬如容易得到高拷贝,在含油酸的培养条件下能够表达膜蛋白等。已有多种外源蛋白在多形汉逊酵母系统中得到表达。本文综述了多形汉逊酵母的基本生物学性质、基础研究领域概况及其在外源基因表达方面的特点和进展。     

Growth simulation of Hansenula polymorpha

Summary Cultivation of Hansenula polymorpha with substrate ethanol in a bench-scale tower loop reactor was simulated by means of a distributed parameter model with regard to the dissolved oxygen and substrate in the medium, oxygen and CO₂ in the gas phase, and a lumped parameter model with regard to the cell mass. Space and time independence of the substrate and oxygen limiting constants of the Monod model, K_S and K_O, was assumed. Time variations of the yield coefficients, Y_X/S and Y_X/O, were allowed for.     

Genetic strains of Hansenula polymorpha

Six centromeric linkage groups and four non-centromeric fragments are revealed in the genetic stocks of Hansenula polymorpha which were obtained by intratetrad breeding in several generations of two genetically different parental strains progeny. Fourteen nuclear markers are mapped, including auxotrophic mutations, mating regulation loci, determinants of sporulation and heat tolerance. Complex origin of the haploid genome of these stocks leads to affinity interactions and to 14 per cent increase in DNA content in haploid stocks, as compared with the parental strains.     

人乳头瘤病毒16亚型L1蛋白在多形汉逊酵母中的优化表达

为了实现人乳头瘤病毒(Humanpa pillomavirus,HPV)16亚型衣壳蛋白L1在多形汉逊酵母(Hansenula polymorpha)中的高效表达,根据L1蛋白的氨基酸序列及多形汉逊酵母的密码子偏爱性,对L1蛋白的编码序列进行优化设计,合成了完整的编码序列,命名为HPV16L1。以甲醇诱导型启动子MOXp和终止子AOXTT为表达调控元件,以尿嘧啶合成相关基因URA3为筛选标记,构建了HPV16L1的重组表达质粒pYMOXU-HPV16。用SacII酶切质粒pYMOXU-HPV16使其线性化,电转化多形汉逊酵母菌株H-ura3,依据营养缺陷互补筛选重组菌株。通过PCR扩增及HPV16L1蛋白表达量分析表明已获得稳定高表达L1蛋白的重组汉逊酵母菌株HP-U-16L。摇瓶发酵条件的初步优化表明,以YPM(pH7.0)为基础培养基进行诱导培养,控制接种量使初始培养液OD600为1.0,每隔12h补加甲醇至终浓度为1%(V/V),37oC、200r/min条件下诱导培养72h后,HPV16L1蛋白的最高表达量为78.6mg/L。本研究为多形汉逊酵母源HPV16L1疫苗的研制奠定了基础。     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

超耐热酸性α-淀粉酶基因在多型汉逊酵母中表达及与在毕赤酵母中表达的比较研究

利用毕赤酵母的质粒载体pPIC9K将极端耐热古菌Pyrococcusfuriosus的超耐热酸性α-淀粉酶(Amy)基因转化到多型汉逊酵母HP-6中,获得重组汉逊酵母。经过甲醇进行诱导,表达产物的酶活性检测和SDS-PAGE电泳,证明α-淀粉酶(Amy)在多型汉逊酵母中利用AOX1启动子和α-因子信号肽有效表达并分泌到胞外。该酶的最适反应温度为90～100℃,最适作用pH为4.0～5.0,较之重组毕赤酵母的最适作用pH还低0.5。此外与毕赤酵母的重组蛋白相比,重组汉逊酵母α-淀粉酶不仅菌株筛选简便、周期短,而且具有更容易筛选到高拷贝转化子以及适用于大规模工业发酵等优点。     

The NII2 gene of Hansenula polymorpha is involved in nitrite assimilation

To establish a basis for genetic and molecular studies of nitrite assimilation in the methylotrophic yeast Hansenula polymorpha, we isolated and characterised six nitrite-negative mutants still capable of growing on nitrate. Gene isolation work yielded the NII2 gene, encoding a membrane protein homologous to the Saccharomyces cerevisiae Pho86p. Sequence analysis revealed an ORF of 860 bp encoding a 286-amino-acid protein with a predicted molecular mass of 32.8 kDa. This protein is shorter than its S. cerevisiae homologue, and is predicted to lack an ER-retention signal. Cell suspension work revealed that the null mutant is unable to take up nitrite from the medium.     

Cloning of maltase gene from a methylotrophic yeast, Hansenula polymorpha

The Hansenula polymorpha maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-alpha-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-alpha-D-glucopyranoside (PNPG), sucrose, maltose and alpha-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-beta-D-galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose 相似文献   

Hansenula polymorpha as a novel yeast system for the expression of heterologous genes

The advantages of Hansenula polymorpha as a new yeast expression system are discussed in terms of the powerful and regulatable methanol oxidase promoter and the organism's ability to grow on cheap carbon sources. The development of techniques for conventional genetic analysis is described. A total of 218 mutants have been assigned to 62 complementation groups, three genes have been found to be linked forming the first linkage group in this organism. Methods for molecular transformation have been developed allowing the expression of heterologous genes. The disruptive integration and expression of the neomycin phosphotransferase is described.     

Simultaneous utilization of heterotrophic substrates by Hansenula polymorpha MH30 results in enhanced growth rates

Summary The maximum specific growth rate (_max) of Hansenula polymorpha MH30 on xylose as the sole source of carbon and energy is 0.175 h^–1, on methanol 0.21 h^–1, on glycerol 0.27 h^–1 and on glucose 0.61 h^–1. On mixtures of xylose plus methanol, xylose plus glycerol, xylose plus glucose and glycerol plus glucose H. polymorpha MH30 grows faster: 0.36 h^–1, 0.37 h^–1, 0.47 h^–1 and 0.52 h^–1, respectively. Attempts have been made to explain these somewhat surprising results, especially the fact that the growth rates on xylose plus methanol and xylose plus glycerol exceed the specific growth rates of these on even the faster partner in the mixture. Offprint requests to: W. Babel