Efeito da adição de oxigênio na produção de glicerol quinase pela levedura recombinante de Pichia pastoris

O glicerol é um importante subproduto de processos fermentativos. Em algumas bebidas alcoólicas, como vinhos e vodcas contribuem com seu sabor tornando-a aceitável para o consumidor. Com o incentivo na produção de biocombustíveis, a disponibilidade do glicerol deve aumentar, já que é o principal subproduto gerado na produção do Biodiesel. A sua presença em alguns alimentos, pode identificar presença de microrganismos, já que pode ser uma fonte de carbono para eles. Devido às inúmeras utilizações do glicerol para diversos tipos de indústrias, a dosagem do glicerol se torna cada vez mais importante e rotineira. Isso incentiva novas pesquisas de métodos de doseamento. A enzima GK é presente em vários microrganismos e converte o glicerol em glicerol-3-fosfato. Com a utilização da enzima Glicerol -3- fofato desidrogenase, o glicerol - 3 – fosfato é convertido a di-hidroxi-acetona fosfato com a produção de NADH. Este pode ser quantificado através do espectrofotômetro, tornando-se um rápido e eficiente método para doseamento. A levedura recombinante Pichia pastoris foi escolhida pois foi inserida no seu DNA, o Gene GUT1 que expressa a GK em Saccharomyces cerevisiae. Além disso, a expressão enzimática é extracelular, diminuindo etapas no processo de isolamento da enzima GK. O trabalho consistiu em otimizar a produção de GK expressa pela levedura recombinante Pichia pastoris por meio da adição de oxigênio no biorreator de fermentação em escala de 2,5 L. Os resultados do presente estudo indicam o ponto ótimo de oxigenação em fermentador nos parâmetros estabelecidos é de 2,0 lpm tanto para a produção de enzimática quanto para biomassa formada

Potential use of bioagents in the control of postharvest rot in melon

Rot caused by Fusarium pallidoroseum has had a severely negative impact on the export of melons from Brazil. Uncertainty regarding the health of the fruit due to the quiescent infection of the pathogen has led producers to use fungicides in the postharvest treatment of the fruit, thereby causing contamination and risking the health of consumers. Consequently, there is a demand for clean and safe natural technologies for the postharvest treatment of melons, including biological control. The present study aimed at evaluating bioagents for use in controlling Fusarium rot in 'Galia'melon. The following bioagents were evaluated: two isolates of Bacillus subtilis, B. licheniformis and a mixture of B. subtilis and B. licheniformis, as well as the yeasts Sporidiobolus pararoseus, Pichia spp., Pichia membranifaciens, P. guilliermondii, Sporobolomyces roseus, Debaryomyces hansenii and Rhodotorula mucilagenosa. Treatment with imazalil and water were used as controls. Two experiments were conducted in a completely randomised design with 10 replicates per treatment with four fruit per replicate; the disease incidence was evaluated in the first experiment, and the disease severity was evaluated in the second. Similarity analysis of the temporal evolution profiles of rot incidence caused by F. pallidoroseum allowed the evaluated treatments to be clustered into four groups. In the first experiment, the yeasts P. membranifaciens and D. hansenii produced results similar to that of the fungicide imazalil. The second experiment highlighted the yeasts P. guilliermondii and R. mucilaginosa. Electron microscopy studies confirmed that once applied to the fruit, the yeasts colonised the skin and damaged the pathogen mycelium; the action of the yeasts affected the mycelium of F. pallidoroseum, which had infected wounds on the fruit's surface. Bacillus spp. did not provide good disease control. These results demonstrated that yeasts have the potential to control postharvest rot caused by F. pallidoroseum in 'Galia'melon.

Using Pichia pastoris to produce recombinant glycerol kinase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de etanol utilizando cascas de banana e de laranja por co-fermentação de Zymomonas mobilis e Pichia stipitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diversity and Physiological Characterization of D-Xylose-Fermenting Yeasts Isolated from the Brazilian Amazonian Forest

Background: This study is the first to investigate the Brazilian Amazonian Forest to identify new D-xylose-fermenting yeasts that might potentially be used in the production of ethanol from sugarcane bagasse hemicellulosic hydrolysates. Methodology/Principal Findings: A total of 224 yeast strains were isolated from rotting wood samples collected in two Amazonian forest reserve sites. These samples were cultured in yeast nitrogen base (YNB)-D-xylose or YNB-xylan media. Candida tropicalis, Asterotremella humicola, Candida boidinii and Debaryomyces hansenii were the most frequently isolated yeasts. Among D-xylose-fermenting yeasts, six strains of Spathaspora passalidarum, two of Scheffersomyces stipitis, and representatives of five new species were identified. The new species included Candida amazonensis of the Scheffersomyces clade and Spathaspora sp. 1, Spathaspora sp. 2, Spathaspora sp. 3, and Candida sp. 1 of the Spathaspora clade. In fermentation assays using D-xylose (50 g/L) culture medium, S. passalidarum strains showed the highest ethanol yields (0.31 g/g to 0.37 g/g) and productivities (0.62 g/L.h to 0.75 g/L.h). Candida amazonensis exhibited a virtually complete D-xylose consumption and the highest xylitol yields (0.55 g/g to 0.59 g/g), with concentrations up to 25.2 g/L. The new Spathaspora species produced ethanol and/or xylitol in different concentrations as the main fermentation products. In sugarcane bagasse hemicellulosic fermentation assays, S. stipitis UFMG-XMD-15.2 generated the highest ethanol yield (0.34 g/g) and productivity (0.2 g/L.h), while the new species Spathaspora sp. 1 UFMG-XMD-16.2 and Spathaspora sp. 2 UFMG-XMD-23.2 were very good xylitol producers. Conclusions/Significance: This study demonstrates the promise of using new D-xylose-fermenting yeast strains from the Brazilian Amazonian Forest for ethanol or xylitol production from sugarcane bagasse hemicellulosic hydrolysates.

Fermentation medium and oxygen transfer conditions that maximize the xylose conversion to ethanol by Pichia stipitis

The xylose conversion to ethanol by Pichia stipitis was studied. In a first step, the necessity of supplementing the fermentation medium with urea. MgSO(4) x 7H(2)O, and/or yeast extract was evaluated through a 2(3) full factorial design. The simultaneous addition of these three nutritional sources to the fermentation medium, in concentrations of 2.3, 1.0, and 3.0 g/l, respectively, showed to be important to improve the ethanol production in detriment of the substrate conversion to cell. In a second stage, fermentation assays performed in a bioreactor under different K(L)a (volumetric oxygen transfer coefficient) conditions made possible understanding the influence of the oxygen transfer on yeast performance, as well as to define the most suitable range of values for an efficient ethanol production. The most promising region to perform this bioconversion process was found to be between 2.3 and 4.9 h(-1), since it promoted the highest ethanol production results with practically exhaustion of the xylose from the medium. These findings contribute for the development of an economical and efficient technology for large scale production of second generation ethanol. (C) 2011 Elsevier Ltd. All rights reserved.

CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis

Abstract Background Fuel ethanol production from sustainable and largely abundant agro-residues such as sugarcane bagasse (SB) provides long term, geopolitical and strategic benefits. Pretreatment of SB is an inevitable process for improved saccharification of cell wall carbohydrates. Recently, ammonium hydroxide-based pretreatment technologies have gained significance as an effective and economical pretreatment strategy. We hypothesized that soaking in concentrated aqueous ammonia-mediated thermochemical pretreatment (SCAA) would overcome the native recalcitrance of SB by enhancing cellulase accessibility of the embedded holocellulosic microfibrils. Results In this study, we designed an experiment considering response surface methodology (Taguchi method, L8 orthogonal array) to optimize sugar recovery from ammonia pretreated sugarcane bagasse (SB) by using the method of soaking in concentrated aqueous ammonia (SCAA-SB). Three independent variables: ammonia concentration, temperature and time, were selected at two levels with center point. The ammonia pretreated bagasse (SCAA-SB) was enzymatically hydrolysed by commercial enzymes (Celluclast 1.5 L and Novozym 188) using 15 FPU/g dry biomass and 17.5 Units of β-glucosidase/g dry biomass at 50°C, 150 rpm for 96 h. A maximum of 28.43 g/l reducing sugars corresponding to 0.57 g sugars/g pretreated bagasse was obtained from the SCAA-SB derived using a 20% v/v ammonia solution, at 70°C for 24 h after enzymatic hydrolysis. Among the tested parameters, pretreatment time showed the maximum influence (p value, 0.053282) while ammonia concentration showed the least influence (p value, 0.612552) on sugar recovery. The changes in the ultra-structure and crystallinity of native SCAA-SB and enzymatically hydrolysed SB were observed by scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The enzymatic hydrolysates and solid SCAA-SB were subjected to ethanol fermentation under separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) by Scheffersomyces (Pichia) stipitis NRRL Y-7124 respectively. Higher ethanol production (10.31 g/l and yield, 0.387 g/g) was obtained through SSF than SHF (3.83 g/l and yield, 0.289 g/g). Conclusions SCAA treatment showed marked lignin removal from SB thus improving the accessibility of cellulases towards holocellulose substrate as evidenced by efficient sugar release. The ultrastructure of SB after SCAA and enzymatic hydrolysis of holocellulose provided insights of the degradation process at the molecular level.

Evaluation of oat hull hemicellulosic hydrolysate fermentability employing Pichia stipitis

Oat hull hemicellulosic hydrolysate obtained by diluted acid hydrolysis was employed as fermentation medium for Pichia stipitis cultivation. A comparison between the use of treated hydrolysate with 1% activated charcoal to reduce the toxic compounds generated during the hydrolysis process and untreated hydrolysate as a control was conducted. In the cultures using treated hydrolysate the total consumption of glucose, low xylose consumption and ethanol and glycerol formation were observed. The medium formulated with untreated hydrolysate showed morphological cell modifications with consequently cell death, no ethanol formation and formation of glycerol as byproduct of fermentative process, probably as a response to stressful conditions to yeast due to presence of high concentration of toxic compounds. Thus, further studies are suggested in order to determine the best conditions for hydrolysis and detoxification of the hydrolysate to improve the fermentative performance of P. stipitis.

Enhancement of heterologous expression of alkaline phytase in Pichia pastors

Phytic acid is the major storage form of phosphorus and inositol in seeds and legumes. It forms insoluble phytate salts by chelating with positively charged mineral ions. Non-ruminant animals are not able to digest phytate due to the lack of phytases in their GI tracks, thus the undigested phytate is excreted leading to environmental contamination. Supplementation with phytases in animal feed has proven to be an effective strategy to alleviate nutritional and environmental issues. The unique catalytic and thermal stability properties of alkaline phytase from lily pollen (LlALP) suggest that it has the potential to be useful as a feed supplement. Our goal is to develop a method for the production of substantial amounts of rLlALP for animal feed and structural studies. rLlALP2 has been successfully expressed in the yeast, Pichia pastoris. However, expression yield was modest (8-10 mg/L). Gene copy number has been identified as an important parameter in enhancing protein yields. Multicopy clones were selected using Zeocin-resistance-based vectors and challenging transformants to high Zeocin levels under different conditions. Data indicate that increasing selection pressure led to the generation of clones with amplification of both rLlAlp2 and Zeor genes and the two genes were not equally amplified. Additionally, clones generated by step-wise methods led to clones with greater amplification. The effects of transgene copy number and gene sequence optimization on expression levels of rLlALP2 were examined. The data indicate that increasing the copy number of rLlAlp2 in transformed clones was detrimental to expression level. The use of a sequence-optimized rLlAlp2 (op-rLlAlp2) increased expression yield of the active enzyme by 25-50%, suggesting that transcription and translation efficiency are not major bottlenecks in the production of rLlALP2. Lowering induction temperature to 20 oC led to an increase in enzyme activity of 1.2 to 20-fold, suggesting that protein folding or post-translational processes may be limiting factors for rLlALP2 production. Cumulatively, optimization of copy number, gene sequence optimization and reduced temperature led to increase of rLlALP2 enzyme activity by three-fold (25-30 mg/L). In an effort to simplify the purification process of rLlALP2, extracellular expression of phytase was investigated. Extracellular expression is dependent on the presence of an appropriate secretion signal upstream of the transgene native signal peptide(s) present in the transgene may also influence secretion efficiency. The data suggest that deletion of both N- and C-terminal signal peptides of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion of LlALP2 by four-fold. The secretion signal peptide of chicken egg white lysozyme was ineffective in secretion rLlALP2 in P. pastoris. To enhance rLlALP2 secretion, effectiveness of the strong inducible promoter (PAOX1) was compared with the constitutive promoter (PGAP). The intracellular yield of rLlALP2 was about four-fold greater under the control of PGAP compared to PAOX1 and extracellular expression level of rLlALP2 was around eight-fold (75-100 mg/L) greater. The successful production of active rLlALP2 in P. pastoris will allow us to conduct the animal feed supplementation studies and structural studies.

Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Human heteromeric amino acid transporters (HATs) play key roles in renal and intestinal re-absorption, cell redox balance and tumor growth. These transporters are composed of a heavy and a light subunit, which are connected by a disulphide bridge. Heavy subunits are the two type II membrane N-glycoproteins rBAT and 4F2hc, while L-type amino acid transporters (LATs) are the light and catalytic subunits of HATs. We tested the expression of human 4F2hc and rBAT as well as seven light subunits in the methylotrophic yeast Pichia pastoris. 4F2hc and the light subunit LAT2 showed the highest expression levels and yields after detergent solubilization. Co-transformation of both subunits in Pichia cells resulted in overexpression of the disulphide bridge-linked 4F2hc/LAT2 heterodimer. Two sequential affinity chromatography steps were applied to purify detergent-solubilized heterodimers yielding ~1mg of HAT from 2l of cell culture. Our results indicate that P. pastoris is a convenient system for the expression and purification of human 4F2hc/LAT2 for structural studies.

Cultivation strategies to enhance productivity of Pichia pastoris: A review.

Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.

Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Δ cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

Pex17p Is Required for Import of Both Peroxisome Membrane and Lumenal Proteins and Interacts with Pex19p and the Peroxisome Targeting Signal–Receptor Docking Complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Δ mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Δ mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal–receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.