Zusammenhang zwischen dem Gärungsverhalten und dem Stickstoffmetabolismus in der Hefe Pichia stipitis

The yeast Pichia stipitis is of biotechnological importance because of her ability to ferment the sugar xylose to ethanol. This is not very widespread among yeast. Xylose can be obtained out if the wood composite hemicellulose, what makes wastes of paper and wood industry a possible source for bioethanol and biofuel. To pave the way to efficient biofuel production the physiological and genetically particularities of P. stipitis need to be better understood Within this work we first tested the ability of Pichia stipitis to use amino acids as carbon and nitrogen source. For this reason we performed growth experiments on several distinct amino acids. The gained results showed that P. stipitis is able to use glutamate, aspartate and proline as sole source of carbon and nitrogen. This is a trait, not very common and not very well examinated in yeasts. Only a few yeasts are known to possess this ability. To see how distributed this ability is, a choice of 34 yeasts strains, belonging to 24 species, was tested. Their growth on glutamate, aspartete and proline was determined. 20 of the tested yeasts were able to use amino acids as carbon source. This ability does not depend on the belonging to a defined cladus. After this observation we took a more detailed look at the behaviour of P. stipitis during growth on glutamate as carbon and as nitrogen source. An interesting result was that the ethanol yield and formation rate was higher when glutamate was used as nitrogen source compared to ammonium. In both cases glucose was used as carbon source. Another result of the experiment was that glutamate was used as carbon source simultaneously with glucose. In this constellation seems to be no carbon catabolite repression caused by glucose. When cultivated with glutamate as carbon source, P. stipitis secretes a great amount of ammonium into the medium which has so far not been described for yeasts. An ammonium secretion seems to point at an efficient ammonium export system. The growth experiments with glutamate as carbon source showed that P. stipitis grows after adaption with a rate similar to that on medium with glucose and glutamate or glucose and ammonium. The biomass yield is also very similar to that in cultivation with glucose, drawn to the yield per mol C-source. To get an insight into the physiological background of the utilization of glutamate as carbon source the activity of the glutamate dehydrogenases GDH1 and GDH2 was examinated. The activity of the catabolic GDH2 was measured whenever glutamate or aspartate were present in the medium. The highest activity was measured with glutamate or aspartate as carbon source. It was observed that the GDH2 activity is increased when ammonium is present in cultures with glutamate or aspartate as carbon source. No activity of the GDH2 was found during cultivation on glucose and ammonium. The activity of the anabolic GHD1 was only found in presence of glucose and ammonium, even if glutamate was also present. The correlation between glutamate utilization and GDH2 activity leads to the idea of disrupting the PsGDH2 gene. For this reason a strain disrupted in the PsKU80 gene was constructed. This was done to circumvent the high rate of non-homologous integration of DNA, transformed into P. stipitis. The disruption of the KU80 gene was successful and the gained mutant showed nearly 100 percent of homologous integration of DNA after transformation. This strain provides a useful tool for further genetic approaches on P. stipitis. Further information about these strain is given in the article send with these synopsis. Using the KU80 strain we performed the disruption of the GDH2 gene in P. stipitis. The gained transformant was unable to use glutamate as carbon or nitrogen source. It was also unable to use aspartate or proline as carbon source. But to a limited amount it was able to use these two amino acids as nitrogen source. A retransformation with an amplificate of the GDH2 ORF restored the ability to utilize glutamate. After these experiments a random mutagenesis was performed to obtain mutants that are unable to utilize glutamate as carbon source. It might have been possible to indentify other factors involved into the regulation of glutamate utilization by these experiments. The random mutagenesis delivered two mutants which were unable to use glutamate as carbon source. But they were still able to use it as nitrogen source. In a plasmid-rescue these mutants were identified as ubiquitin ligase and cytochrome-C-oxidase subunit V (COX5). The COX5 mutant was chosen for further characterization. This was done by determination of the substrate spectrum, measuring of the ethanol formation under different aeration conditions, and by testing the sensitivity to inhibitors of the respiratory chain. The COX5 mutant was unable to utilize any kind of non-fermentable carbon source. No activity of cytochrome-C-oxidase was detectable in crude cell extracts of the mutant. According to these observations it seems likely that the respiratory chain is no longer able supply energy for the cells. The COX 5 mutant forms significantly more ethanol under fully aerated conditions, compared to wild type and transformation strain. The ethanol formation is also higher on glucose compared to xylose. Under less aerated conditions the ethanol yield of the COX5 mutant was lower but still higher than in the wild type. These findings lead to the estimation that in the COX5 mutant the fermentative pathways are more active than in the wild type under highly aerated conditions. Growth experiments with SHAM, the inhibitor of the alternative oxidase, showed that the COX5 mutant is completely sensitive to SHAM. The gas exchange was affected by SHAM in the same way. The wild type shows only minor reactions to high SHAM concentrations. This sensitivity of the mutant to SHAM shows the importance of the alternative oxidase after the lost of cytochrome-C-oxidase activity. The role seems to lie in maintaining the redox-balance of the cell, not in energy supply. If the alternative oxidase would deliver energy the mutant should be able to grow on non-fermentable substrates. Cultivations with KCN reduced the drymass yield of the wild type, but increased the one of the mutant. These observations were also supported by measuring the gas exchange. The gas exchange of the mutant was also increased in presence of KCN. Concerning these findings a stimulation effect of the KCN on the alternative oxidase could be possible.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2009

Author dc:creator

• Freese, Stefan

Contributors dc:contributor

• Klinner, Ulrich

• info:eu-repo/classification/ddc/570
• Hefeartige Pilze
• Bioalkohol
• Glutamatdehydrogenase
• Biowissenschaften, Biologie
• Hefe
• Bioethanol
• Ethanolbildung
• Cytrochromeoxidase
• C-assimilatorische Aminosäurenutzung
• Pichia stipitis
• yeast
• glutamate dehydrogenase
• biofuel
• cytochrome
• amino acids

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-113590%22

OAI record oai:identifier

https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51283