The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from theTrichoderma reeseiCel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The twoT. reeseiCBM1s as well as the CBM3 from theClostridium thermocellumCipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face ofValoniacellulose crystals. The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis. The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out inPichia pastoris. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, theT. reeseiCel6A CBM1 was expressed on the surface of thegram-positive bacteria,Staphylococcus carnosus. The engineeredS. carnosuscells were shown to bind cellulosefibers. To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of theS. carnosusand clearly enhanced the metal bindingability of the engineered bacteria. <b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,Trichoderma reesei, Staphyloccus carnosus.

<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>

With the introduction of antibiotics in animal feed becoming less popular, the agricultural industry has begun a shift towards the use of probiotics in animal feed. Since there is no current method to evaluate the risks of using genetically modified probiotics in animal feed. The goal of this project was to create a genetically modified model organism for risk assessment. The genetic marker for that was chosen was GFP that was to be expressed on the surface of the cell. The fluorescent properties allow for visualisation of the genetically modified bacteria and the surface expression would allow for the easy capture and recovery of the bacteria for culturing and cell counts. Genome wide screens were performed using the CW PRED algorithm to locate proteins with LPXTG motif for cell wall anchoring. 16 hypothetical proteins were detected and 6 were selected as candidates for possible surface display of GFP. Of these candidates, the novel L. casei protein LSEI_2320 was found to be expressed at the mRNA during early growth by RT PCR and at then protein level during stationary phase with western blot. This LPXTG protein was found at the surface of L. casei ATCC334 during stationary phase and late stationary phase with immunofluorescence microscopy. A genetically modified L. casei ATCC334 was constructed using the surface protein LSEI_2320 locus as a region for recombination with the pRV300 suicide plasmid. Genetic modification of the locus by the insertion of a GFP reporter region just before the predicted signal peptide site resulted in the abrogation of the expression of LSEI_2320 from the cell surface at the late stationary phase. It appears that this particular gene is not necessary to cell survival even though it is abundantly expressed on the cell surface and can be used as a location for genetic modification in L. casei ATCC334.

With the introduction of antibiotics in animal feed becoming less popular, the agricultural industry has begun a shift towards the use of probiotics in animal feed. Since there is no current method to evaluate the risks of using genetically modified probiotics in animal feed. The goal of this project was to create a genetically modified model organism for risk assessment. The genetic marker for that was chosen was GFP that was to be expressed on the surface of the cell. The fluorescent properties allow for visualisation of the genetically modified bacteria and the surface expression would allow for the easy capture and recovery of the bacteria for culturing and cell counts. Genome wide screens were performed using the CW PRED algorithm to locate proteins with LPXTG motif for cell wall anchoring. 16 hypothetical proteins were detected and 6 were selected as candidates for possible surface display of GFP. Of these candidates, the novel L. casei protein LSEI_2320 was found to be expressed at the mRNA during early growth by RT PCR and at then protein level during stationary phase with western blot. This LPXTG protein was found at the surface of L. casei ATCC334 during stationary phase and late stationary phase with immunofluorescence microscopy. A genetically modified L. casei ATCC334 was constructed using the surface protein LSEI_2320 locus as a region for recombination with the pRV300 suicide plasmid. Genetic modification of the locus by the insertion of a GFP reporter region just before the predicted signal peptide site resulted in the abrogation of the expression of LSEI_2320 from the cell surface at the late stationary phase. It appears that this particular gene is not necessary to cell survival even though it is abundantly expressed on the cell surface and can be used as a location for genetic modification in L. casei ATCC334.

This dissertation introduces FESM (Flood Event Simulation Model), a Geographic Information System (GIS) tool designed for use on gaged river systems that can be used to guide logistic support during disaster events. FESM rapidly generates flood predictions using elevation data from real-world sensors or generated by other models. Verification and validation data for FESM are provided. In order to construct a visualization system for interacting with FESM outputs, single buffer and dual buffer techniques for moving massive datasets to the GPU for processing using OpenCL were rigorously tested and timed, and an analysis of the costs/benefits of using buffers or images was conducted. Finally, DRO (Dynamic Raster Overlay), a visualization system for analysis of datasets composed of multiple overlapping flood maps is introduced, and expert feedback is provided on the effectiveness of DRO with selected case studies.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25398号 / エネ博第477号 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 准教授 中田 栄司, 教授 菅瀬 謙治 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications. Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells. Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions. Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes. The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications. The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated. <b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

The versatile Escherichia coli facilitates protein expression with relative simplicity, high cell density on inexpensive substrates, well known genetics, variety of expression vectors, mutant strains, co-overexpression technology, extracytoplasmic secretion systems, and recombinant protein fusion partners. Although, the protocol is rather simple for soluble proteins, heterologous protein expression is frequently encountered by major technical limitations including inefficient translation, formation of insoluble inclusion bodies, lack of posttranslational modification mechanisms, degradation by host proteases, and impaired cell physiology due to host/protein toxicity, in achieving functional expression of stable, soluble, and bioactive protein.. In this thesis, model protein expression systems are used to address the technical issues for enhancing recombinant protein expression in E. coli. When yellow fluorescence protein (YFP) was displayed on E. coli cell surface, the integrity of the cell envelope was compromised and cell physiology was severely impaired, resulting in poor display performance, which was restored by the coexpression of Skp, a periplasmic chaperone. On the basis of monitoring the promoter activities of degP, rpoH, and cpxP under various culture conditions, it was demonstrated that the cell-surface display induced the σE extracytoplasmic stress response, and PdegP::lacZ was proposed to be a suitable “sensor” for monitoring extracytoplasmic stress. Intracellular proteolysis has been recognized as one of the key factors limiting recombinant protein production, particularly for eukaryotic proteins heterologously expressed in the prokaryotic expression systems of E. coli. Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression. The study offers an alternative genetic strategy in molecular manipulation to enhance recombinant protein production in E. coli. To overcome the technical limitations of protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis in the functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli, an alternative approach was explored by extracellular secretion of PalB via two Sec-independent secretion systems, i.e. the α-hemolysin (Type I) and the modified flagellar (Type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Bioactive PalB was expressed and secreted extracellularly either as HlyA fusion (i.e. PalB-HlyA via Type I system) or an intact protein (via Type III system) with minimum impact on cell physiology. However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. PalB secretion via Type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via Type III system was slow with lower specific PalB activities but effective cell growth. Functional expression of lipase from Burkholderia sp. C20 (Lip) in various cellular compartments of Escherichia coli was explored. The poor expression in the cytoplasm was improved by several strategies, including coexpression of the cytoplasmic chaperone GroEL/ES, using a mutant E. coli host strain with an oxidative cytoplasm, and protein fusion technology. Fusing Lip with the N-terminal peptide tags of T7PK, DsbA, and DsbC was effective in boosting the solubility and biological activity. Non-fused Lip or Lip fusions heterologously expressed in the periplasm formed insoluble aggregates with a minimum activity. Biologically active and intact Lip was obtained upon the secretion into the extracellular medium using the native signal peptide and the expression performance was further improved by coexpression of the periplasmic chaperon Skp. The extracellular expression was even more effective when Lip was secreted as a Lip-HlyA fusion via the α-hemolysin transporter. Finally, Lip could be functionally displayed on the E. coli cell surface when fused with the carrier EstA.

<p>The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.</p><p>Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.</p><p>Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.</p><p>Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.</p><p>The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.</p><p>The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.</p><p><b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices</p>