Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.     

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

A collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl-CoA synthetase (acs1, acs2 and acs3). All mutations except acs1-acs3 have no effect on the activities of other enzymes involved in C₂ metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol-induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C₂ compound-induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.     

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica. The P. methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80-88%). In P. methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose. Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium. These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth.     

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.     

Identification of major ADH genes in ethanol metabolism of Pichia pastoris

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.     

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.     

Development of the methylotrophic yeast Pichia methanolica for the expression of the 65 kilodalton isoform of human glutamate decarboxylase

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD₆₅) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD₆₅ at levels approaching 0·5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD₆₅ was purified to greater than 90% purity. © 1998 John Wiley & Sons, Ltd.     

Linear DNA plasmid pPK2 of Pichia kluyveri: distinction between cytoplasmic and mitochondrial linear plasmids in yeasts

The linear plasmids frequently found in plants and filamentous fungi are associated with mitochondria or chloroplasts. In contrast, all the linear plasmids known in yeasts are cytoplasmic elements. From a strain of the yeast Pichia kluyveri, we have isolated a new linear plasmid, pPK2, which was found to be associated with mitochondria. This 7·1 kilobase pairs‐long DNA contained only two genes, which code for DNA and RNA polymerases, as judged from their nucleotide sequences translated by a mitochondrial genetic code. When we examined several recently isolated yeast plasmids for their subcellular localization, we found that two linear plasmids, pPH1 from Pichia heedii, as well as pPK1 from another strain of P. kluyveri, were also localized in mitochondria. These plasmids are the first examples of mitochondria‐associated linear plasmids in yeast. All other linear plasmids we examined were of cytoplasmic origin. Whilst the cytoplasmic type linear plasmids were efficiently eliminated by ultraviolet irradiation of host cells, the mitochondria‐associated plasmids were highly resistant. The mitochondrial pPK2 plasmid was rapidly lost by treatment of the host cells with ethidum bromide. Copyright © 1999 John Wiley & Sons, Ltd.     

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris

Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirDeltakex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirDeltakex1 while it was only 1.1 g/l in GS115Hir.     

季也蒙毕赤酵母Y35-1菌株对枇杷采后炭疽病的抑菌效果及保鲜作用

利用从原生态枇杷果实上分离筛选并鉴定的、能显著抑制炭疽病的季也蒙毕赤酵母Y35-1菌株,研究其抑菌抗腐效果,并将其制成液体和固体型菌剂在枇杷贮藏保鲜方面应用。结果表明：拮抗酵母Y35-1菌株能够有效抑制枇杷炭疽病胶孢炭疽菌孢子的萌发,能够在病原菌菌丝上寄生而抑制菌丝的正常生长,不接触对峙培养实验显示Y35-1菌株能够有效抑制病原菌菌丝的扩展和生长。保鲜应用表明：酵母Y35-1液体和活性冻干粉菌剂均能明显抑制枇杷果实贮藏期间的病害腐烂发生率,贮藏至第20天时,两处理组果实腐烂指数分别仅为2.25%和3.60%;且这两种生防菌剂对枇杷的硬度、质量损失率、可溶性固形物、果皮细胞膜透性、总糖含量、总酸含量、VC含量均起到了一定的维持作用。Y35-1酵母液体菌剂在4?℃条件下、活性冻干粉菌剂在－20?℃条件下最适宜存放,且海藻糖对Y35-1酵母贮藏期间生活力和生防效力起到了保护作用。研究结果显示Y35-1菌株对采后枇杷胶孢炭疽菌有明显的抑菌作用,液体和固体剂型的生防制剂均对采后枇杷具有抑腐保鲜效果。     

果胶多糖水热法降解及其产物体外抗氧化性评价

采用水热法降解商品果胶多糖,并对其降解产物的抗氧化活性进行评价。结果表明,水热法降解果胶多糖的最优工艺条件为水热处理温度140 ℃、水热处理时间30 min、pH 6;在此条件下,果胶多糖降解产物得率达46.2%。在此基础上,采用乙醇分级沉淀法对果胶多糖水热处理液进行分离,得到3 种不同分子质量范围的果胶多糖降解产物（S1、S2和S3）,其重均分子质量分别为13.4、7.5 kDa和5.7 kDa。以商品果胶多糖和3 种降解产物为研究对象,进行抗氧化性评价,结果表明,S1组分对1,1-二苯基-2-苦基肼自由基的清除率达49.8%,是商品果胶的4 倍;S3组分对超氧阴离子自由基的清除率达58.7%,是商品果胶的10 倍。说明水热降解果胶多糖可显著提高其抗氧化活性,为果渣废弃物的高效利用提供理论依据。     

Co-consumption of sugars or ethanol and glucose in a Saccharomyces cerevisiae strain deleted in the HXK2 gene

In previous studies it was shown that deletion of the HXK2 gene in Saccharomyces cerevisiae yields a strain that hardly produces ethanol and grows almost exclusively oxidatively in the presence of abundant glucose. This paper reports on physiological studies on the hxk2 deletion strain on mixtures of glucose/sucrose, glucose/galactose, glucose/maltose and glucose/ethanol in aerobic batch cultures. The hxk2 deletion strain co-consumed galactose and sucrose, together with glucose. In addition, co-consumption of glucose and ethanol was observed during the early exponential growth phase. In S.cerevisiae, co-consumption of ethanol and glucose (in the presence of abundant glucose) has never been reported before. The specific respiration rate of the hxk2 deletion strain growing on the glucose/ethanol mixture was 900 micromol.min(-1).(g protein)(-1), which is four to five times higher than that of the hxk2 deletion strain growing oxidatively on glucose, three times higher than its parent growing on ethanol (when respiration is fully derepressed) and is almost 10 times higher than its parent growing on glucose (when respiration is repressed). This indicates that the hxk2 deletion strain has a strongly enhanced oxidative capacity when grown on a mixture of glucose and ethanol.     

Evaluation of Brazilian ethanol production yeasts for maltose fermentation in media containing structurally complex nitrogen sources

Maltose and glucose fermentations are strongly affected by the structural complexity of the nitrogen source and by the presence of oxygen. In this study five industrial Saccharomyces cerevisiae strains were grown in synthetic medium, containing maltose or glucose, supplemented with different nitrogen sources, with or without agitation. All strains were able to grow and efficiently ferment glucose, but not all strains were able to grow and ferment maltose well. Peptone and ammonium sulfate induced improved fermentation for all strains and conditions. Under agitation, as expected, higher biomass accumulation was detected. Casamino acids supplementation induced efficient maltose fermentation for all of the strains under aerated conditions, but differing maltose utilization patterns were observed for the static cultures. Copyright © 2012 The Institute of Brewing & Distilling