Genome-scale metabolic reconstructions of Pichia stipitis and Pichia pastoris and in-silico evaluation of their potentials (original) (raw)
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Microbial Cell Factories, 2010
Background: Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.
Engineering yeasts for xylose metabolism
Current Opinion in Biotechnology, 2006
Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.
Biotechnology Journal, 2010
The methylotrophic yeast Pichia pastoris has gained much attention during the last decade as a platform for producing heterologous recombinant proteins of pharmaceutical importance, due to its ability to reproduce post-translational modification similar to higher eukaryotes. With the recent release of the full genome sequence for P. pastoris, in-depth study of its functions has become feasible. Here we present the first reconstruction of the genome-scale metabolic model of the eukaryote P. pastoris type strain DSMZ 70382, PpaMBEL1254, consisting of 1254 metabolic reactions and 1147 metabolites compartmentalized into eight different regions to represent organelles. Additionally, equations describing the production of two heterologous proteins, human serum albumin and human superoxide dismutase, were incorporated. The protein-producing model versions of PpaMBEL1254 were then analyzed to examine the impact on oxygen limitation on protein production.
Metabolic engineering of yeast
2000
fermentation. The increasing demand for ethanol as a substitute for gasoline requires the development of lower-cost feedstocks like lignocellulosic ones, which are sufficient to substitute for corn starch (Hacking et al., 1986). Genetic engineering of S. cerevisiae for pentose utilization is an important research target for this purpose. The familiarity and experience of the corn processing industry with yeast fermentations and the potential robustness of the organism makes S. cerevisiae most attractive to the corn processing industry (Bothast et al, 1999). When yeasts are used in these biotechnological processes, however, they have to endure various environmental stresses like nutrient limitation, elevated temperatures and oxidation. Since they respond to these stresses by signal transduction, transcriptional and post-translational control, protein-targeting to organelles and activation of repair functions, which are all energy-requiring cellular activities, they have higher energy requirements under stress conditions (Attfield, 1997). As a consequence, cells must use ATP to expel protons via plasma membrane H'-ATPase activity. Cell survival might therefore be determined by the balance between maintenance of intracellular ATP levels for repair, growth and ATP expenditure for pH homeostasis. The heterologous expression of the bacterial hemoglobin (VHb) in S. cerevisiae is a successful example of metabolic engineering to improve yeast energetics. Upon growth on acetaldehyde, there has been a 3-fold increase in the final cell density of the VHbexpressing S. cerevisiae compared to its control (Chen et al., 1994). Another potentially relevant target for improving energy metabolism is creatine kinase (CK), a key enzyme in energy metabolism of excitable cells and tissues of vertebrate species, that appears to play a complex, multifaceted role in energy homeostasis (WaUimann et al., 1992). As a main function, it catalyzes the reversible phosphorylation from ATP to phosphocreatine (PCr), which serves as an energy-storage metabolite. Thus, the readily accessible phosphorylation potential of PCr can be used to regenerate ATP, by functioning as a temporal energy buffer. The CK system also functions as an energy transport system by connecting sites of energy production with sites of energy utilization and thus maintains a high intracellular ATP-to-ADP ratio. Additionally, as the CK reaction in the direction of ATP synthesis utilizes one proton, it serves as a pH-buffering function, thus counteracting the intracellular acidification. Therefore, the installation of a CK-PCr circuit in yeast seems to have potential to be beneficial to energy metabolism, either by improving overall efficiency, or by the 'energy buffer' function during transient stress conditions. Transient stress conditions occur very commonly in industrial fed-batch cultivations of S. cerevisiae. A typical example is short-term glucose fluctuations during large-scale fed-batch cultivations (Neubauer et al., 1995a). Since these short-term stresses influence cellular physiology and growth, their reduction is crucial for the process 12 performance (Neubauer et al, 1995b). The investigation of the CK-PCr circuit in yeast are reported in Chapter 2. METABOLIC ENGINEERING OF YEAST CENTRAL CARBON METABOLISM Studies on Xylose Utilization and Pentose Phosphate Pathway In addition to yeast energy metabolism, central carbon metabolism is also an important target for metabolic engineering, because it will eventually limit production, once the biosynthetic branches are optimized. Several groups have been working on the improvement of yeast central carbon metabolism for the production of various compounds. Some efforts are directed towards introduction of features from other yeasts, mostly utilization of certain sugars. It is known, for example, that the yeast Pichia stipitis can grow on pentoses like xylose. S. cerevisiae, however, cannot utilize xylose (Walfridsson et al., 1995). However, for the production of fuel ethanol from lower-cost feedstocks, which are the lignocellulosic ones, utilization of pentose sugars like xylose is necessary. Although P. stipitis can grow on xylose, it cannot produce ethanol. Xylose is derived from the hemicellulose component of lignocelluloses (Bothast et al., 1999). For the purpose of xylose utilization, S. cerevisiae was metabolically engineered by cloning the P. stipitis genes XYLI and XYL2 encoding xylose reductase and xylitol dehydrogenase. These two enzymes catalyze the initial steps in xylose utilization, which do not exist in wild type S. cerevisiae. Additionally, the S. cerevisiae TKL1 and TALI genes encoding transketolase and transaldolase were overexpressed, to improve pentose phosphate pathway utilization. The results showed that XYLI-and X)/L2-expressing S. cerevisiae that co-overexpresses TALI had considerably enhanced growth on xylose compared to a strain expressing only XYLI and XYL2. From these data, it was concluded that the insufficient transaldolase levels cause the inefficient utilization of pentose phosphate pathway in S. cerevisiae (Walfridsson et al., 1995). Some aspects of this problem are discussed in Chapter 3. Studies on Glycolysis As the central pathway of carbon metabolism, glycolysis is the prime route to the most important yeast product, ethanol. There have been several attempts to metabolically engineer yeast glycolysis by modifying various enzymes, especially those known as 'rate limiting'. Atypical example is overexpression of eight different enzymes of glycolysis and alcoholic fermentation in S. cerevisiae including hexokinase, phospholructokinase and pyruvate kinase, which catalyze irreversible reactions (Schaaff et al., 1989). However, this overexpression had no effect on ethanol production rate. Additionally, simultaneous increases in the activities of enzyme pairs like pyruvate kinase and phospholructokinase, or pyruvate decarboxylase and alcohol dehydrogenase, did
PLOS ONE, 2016
Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented.
Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis
Nature Biotechnology, 2007
Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose metabolism in Saccharomyces cerevisiae. We have sequenced and assembled the complete genome of P. stipitis. The sequence data have revealed unusual aspects of genome organization, numerous genes for bioconversion, a preliminary insight into regulation of central metabolic pathways and several examples of colocalized genes with related functions. The genome sequence provides insight into how P. stipitis regulates its redox balance while very efficiently fermenting xylose under microaerobic conditions.
Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris
BMC systems biology, 2017
Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions. In this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a ...
Applied Microbiology and Biotechnology, 2014
Xylitol is industrially synthesized by chemical reduction of D-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce D-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of D-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the Dxylulose-forming D-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a singlefermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078 g xylitol/g glucose and productivity of 0.29 g xylitol/ L h. This was the first report to convert xylitol from glucose by the pathway of glucose-D-arabitol-D-xylulose-xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.
Engineering strategy of yeast metabolism for higher alcohol production
Microbial Cell Factories, 2011
Background: While Saccharomyces cerevisiae is a promising host for cost-effective biorefinary processes due to its tolerance to various stresses during fermentation, the metabolically engineered S. cerevisiae strains exhibited rather limited production of higher alcohols than that of Escherichia coli. Since the structure of the central metabolism of S. cerevisiae is distinct from that of E. coli, there might be a problem in the structure of the central metabolism of S. cerevisiae. In this study, the potential production of higher alcohols by S. cerevisiae is compared to that of E. coli by employing metabolic simulation techniques. Based on the simulation results, novel metabolic engineering strategies for improving higher alcohol production by S. cerevisiae were investigated by in silico modifications of the metabolic models of S. cerevisiae.