共查询到20条相似文献,搜索用时 15 毫秒
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Krasovska OS Stasyk OG Nahorny VO Stasyk OV Granovski N Kordium VA Vozianov OF Sibirny AA 《Biotechnology and bioengineering》2007,97(4):858-870
The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system. 相似文献
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G.J. Sulter I.J. van der Klei J.P. Schanstra W. Harder M. Veenhuis 《FEMS microbiology letters》1991,82(3):297-302
Abstract: This paper describes ethanol metabolism in a peroxisome-deficient (PER) mutant of Hansenula polymorpha . The PER mutant was able to use ethanol as sole-carbon source but showed reduced growth rates compared to wild-type cells together with a reduced rate of ethanol utilization under μmax conditions. In chemostat cultures at low-dilution rates, the activities of alcohol dehydrogenase, isocitrate lyase and malate synthase were comparable in wild-type and PER cells. In PER cells the two latter enzymes, exclusively microbody-bound in wild-type cells, were active in the cytosol. The possible advantage of intact microbodies in the intermediary metabolism of ethanol in H. polymorpha is discussed. 相似文献
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Effects of methanol on expression of an anticoagulant hirudin in recombinant Hansenula polymorpha 总被引:1,自引:0,他引:1
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 相似文献
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Isolation of mutants of Hansenula polymorpha defective in the assembly of octameric alcohol oxidase 总被引:1,自引:0,他引:1
Alcohol oxidase (AO) is a peroxisomal enzyme that catalyses the first step in methanol metabolism in yeast. Monomeric, inactive AO protein is synthesised in the cytosol and subsequently imported into peroxisomes, where the enzymatically active, homo-octameric form is found. The mechanisms involved in AO octamer assembly are largely unclear. Here we describe the isolation of Hansenula polymorpha mutants specifically affected in AO assembly. These mutants are unable to grow on methanol and display reduced AO activities. Based on their phenotypes, three major classes of mutants were isolated. Three additional mutants were isolated that each displayed a unique phenotype. Complementation analysis revealed that the isolated AO assembly mutants belonged to 10 complementation groups. 相似文献
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The GSH2 gene, encoding Hansenula polymorpha gamma-glutamylcysteine synthetase, was cloned by functional complementation of a glutathione (GSH)-deficient gsh2 mutant of H. polymorpha. The gene was isolated as a 4.3-kb XbaI fragment that was capable of restoring GSH synthesis, heavy-metal resistance and cell proliferation when introduced into gsh2 mutant cells. It possesses 53% identical and 69% similar amino acids compared with the Candida albicans homologue (Gcs1p). In comparison to the Saccharomyces cerevisiae homologue (Gsh1p), it possesses 47% identical and 61% similar amino acids. The GSH2 sequence appears in the GenBank database under accession No. AF435121. 相似文献
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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 总被引:1,自引:0,他引:1
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。 相似文献
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Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures 总被引:1,自引:0,他引:1
Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol l(-1), which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid l(-1), reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply. 相似文献
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Kirsten Kottmeier Jost Weber Carsten Müller Thomas Bley Jochen Büchs 《Biotechnology and bioengineering》2009,104(3):554-561
Hansenula polymorpha RB11 pC10‐FMD (PFMD− GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system “BioLector” under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation. Biotechnol. Bioeng. 2009; 104: 554–561 © 2009 Wiley Periodicals, Inc. 相似文献
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Oh DB Park JS Kim MW Cheon SA Kim EJ Moon HY Kwon O Rhee SK Kang HA 《Biotechnology journal》2008,3(5):659-668
The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans. 相似文献
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Stasyk OV Nazarko VY Pochapinsky OD Nazarko TY Veenhuis M Sibirny AA 《FEMS yeast research》2003,4(2):141-147
Two interacting AAA ATPases, Pex1p and Pex6p, are indispensable for peroxisome biogenesis in different organisms. Mutations affecting corresponding genes are the most common cause of the peroxisome biogenesis disorders in humans. By UV mutagenesis of the Hansenula polymorpha pex6 mutant, deficient in peroxisome biogenesis, we isolated a conditional cold-sensitive strain with restored ability to grow in methanol medium at 37 degrees C but not at 28 degrees C. Sequencing of the pex6 allele revealed a point mutation in the first AAA module of the PEX6 gene that leads to substitution of a conserved amino acid residue (G737E). An additional intragenic mutation identified in the cold-sensitive pex6 allele leads to a conserved amino acid substitution in the second AAA domain (R1000G). Electron microscopic analysis revealed restored peroxisomes in methanol-induced cold-sensitive pex6 cells at both permissive and restrictive temperatures. If separated, the secondary mutation did not affect methylotrophic growth. Our data suggest that H. polymorpha Pex6p may have a complex function in peroxisome biogenesis in which identified amino acid residues are involved. 相似文献
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Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin 总被引:1,自引:0,他引:1
Several yeast systems have recently been developed for the recombinant production of gelatin and collagen. Amino acid sequence-specific prolyl 4-hydroxylation is essential for the gel-forming capacity of gelatin and for the proper folding of (pro)collagen. This post-translational modification is generally considered to be absent in microbial eukaryotic systems and therefore co-expression of heterologous (human or animal) prolyl 4-hydroxylase would be required. However, we found that the well-known protein expression host Hansenula polymorpha unexpectedly does have the endogenous capacity for prolyl 4-hydroxylation. Without co-expression of a heterologous prolyl 4-hydroxylase, both an endogenous collagen-like protein and a heterologously expressed collagen fragment were found to be sequence-specifically hydroxylated. 相似文献
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Agaphonov M Romanova N Sokolov S Iline A Kalebina T Gellissen G Ter-Avanesyan M 《FEMS yeast research》2005,5(11):1029-1035
Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing. 相似文献
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The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase was investigated in the methanol-utilizing yeast Hansenula polymorpha. The organism was found to synthesize immunologically identical alcohol oxidases during growth on glycerol and methanol. Growth on glycerol, however, was not dependent on the alcohol oxidase, as was shown with a mutant without alcohol oxidase protein. Similarly it was shown with a catalase activity negative mutant that high catalase activity during growth on glycerol was not a prerequisite for the utilization of this substrate, though absolutely required for growth on methanol.Experiments were conducted with mixed substrates to study the influence of methanol on alcohol oxidase synthesis. In batch cultures, growth on ribose plus methanol resulted in an enhanced rate of alcohol oxidase synthesis as compared to ribose alone. In continuous cultures, (D=0.1 h-1) addition of methanol to glycerol-, glucose-, or sorbose-limited cultures gave rise to increased alcohol oxidase activity of up to 20 U/mg, which is about by 2 times higher than the specific activity used for growth on methanol alone. The increase in specific activity of the dissimilatory enzymes on the mixed substrates is partly due to methanol per se, as was shown by a mutant unable to dissimilate or assimilate methanol. 相似文献
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Degelmann A Müller F Sieber H Jenzelewski V Suckow M Strasser AW Gellissen G 《FEMS yeast research》2002,2(3):349-361
The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media. 相似文献
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Ubiyvovk VM Blazhenko OV Gigot D Penninckx M Sibirny AA 《Cell biology international》2006,30(8):665-671
GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding gamma-glutamyltranspeptidase (gammaGT). This is confirmed by the absence of the corresponding activity of gammaGT in the mutant with disrupted GGT1 gene. It was shown that gammaGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a gammaGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products. 相似文献
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C H Kim H W Seo H Y Kim J H Sohn E S Choi S K Rhee 《Journal of industrial microbiology & biotechnology》1998,21(1-2):1-5
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 相似文献
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Roman Jansen Holger Morschett Dennis Hasenklever Matthias Moch Wolfgang Wiechert Marco Oldiges 《Biotechnology progress》2021,37(4):e3144
In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor. 相似文献