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Designer cellulosome for application to advanced biotechnology tools
S68
Abstracts / New Biotechnology 33S (2016) S1–S213
cassette. Since a crucial step for cell wall localization of the enzymes is the secretion of proteins in yeast cells, we also demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the SED1 gene for cell-surface display of cellulolytic enzymes. http://dx.doi.org/10.1016/j.nbt.2016.06.956
S1-2 Designer cellulosome for application to advanced biotechnology tools Sung Ok Han ∗ , Jeong Eun Hyeon, Seung Kyou You Korea University, Republic of Korea The cellulosome is one of nature’s most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. First, to construct the self-assembled biosensor with signal amplification, a cellulosome system, comprising Type I and Type II dockerin–cohesin interactions with different specificity, from the anaerobic Clostridia bacterium was applied. The self-assembled biosensor was highly sensitive and achieved 128.1fold increase in detection levels compared to the control. Second, the designer enzymes and microbes are a key biological technology that can be used for biorefinery. Prior to fermentative production of valuable product from the biomass by microbes, the polymeric substrates are hydrolyzed to release monomeric sugars. The assembly of minicellulosomes by Saccharomyces cerevisiae and Corynebacterium glutamicum increased the activity against various lignocellulosic materials by approximately 3-fold compared with control. Also, red algae-degrading complexes was produced by Escherichia coli and increased the activity against the marine biomass substrate by approximately 2-fold, compared with that for the corresponding enzymes alone. Final, enzyme complex for C1 gas conversion was designed. Carbon monoxide (CO) was successfully efficiently converted by functional complexes containing carbon monoxide dehydrogenase (CODH) and carbon monoxide sensing heme protein (CooA) with enhanced CO binding affinity. An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of CO2 -utilizing Ralstonia eutropha and successfully showed 3.3-fold increased conversion efficiency. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. http://dx.doi.org/10.1016/j.nbt.2016.06.957
S1-3 Hybrid library
nanocellulosome-chimera
designed
from
module
Mitsuo Umetsu Tohoku University, Japan Structural and functional information of proteins are stored by the unit of domain (module) which can be expressed in E. coli, and molecular evolutional techniques prefer to functionalize proteins by the units. Further, in the field of materials, “Nanotechnology” came in use from 1980s, so that various organic and inorganic materials are downsized to nano-meter which is comparable to the size
of the protein domain and modules. In the “smart nano-bio design” we propose, the protein module and various organic/inorganic nanomaterials are considered as “functional nanomaterials” with no distinction of matters, and they are constructively built up in vitro on a scaffold structure of organic or inorganic nanomaterial without being bound to the limitation of structural design using amino acid sequence. In the environmental field, we focus on the enzyme of cellulase. Cellulases are generally modular proteins with independent catalytic and cellulose-binding domains (CBDs), and in some bacteria, catalytic domains (CDs) are non-covalently assembled on a scaffold protein with CBD to form a giant protein complex, called a cellulosome, which efficiently degrades water-insoluble hard materials. Recently, we proposed a new design for artificial cellulosomes with multiple CBDs on non-cellulosome-derived scaffold structures. CDs and CBDs are independently prepared by recombinant means, and they are heterogeneously clustered on inorganic nanoparticle. The heteroclustering of CD with CBD on nanomaterials resulted in significant improvements in the enzyme’s degradation activity for water-insoluble substrates. Here, we show the potential of the combination of nanomaterials and protein module for designing the cellulase with high performance. http://dx.doi.org/10.1016/j.nbt.2016.06.958
S1-4 Overproduction of cellulase by Trichoderma reesei Rut C30 through batch-feeding of synthesized mixture as substrate and inducer Feng-Wu Bai Shanghai Jiao Tong University, China Cellulase for hydrolyzing cellulose in lignocellulosic biomass to release glucose is a prerequisite for producing biofuels and biobased chemicals through microbial fermentation (biorefinery), but high cost with cellulase presents one of the biggest challenges. Here we report the synthesis of low-cost mixture from glucose by -glucosidase through the transglycosylation reaction as substrate for the growth of Trichoderma reesei Rut C30 and inducer for cellulase production. Compared to commonly used soluble inducer lactose, the sugar mixture facilitated mycelial growth, and induced cellulase production more effectively due to the presence of easily assimilated glucose as carbon source for mycelial growth and betadisaccharides, in particular sophorose, for cellulase induction. As a result, cellulase activity as high as 90.3 FPU (filter paper unit)/mL was achieved at 144 h with the fed-batch strategy through which glucose was controlled between 0.05 g/L and 0.30 g/L to eliminate its inhibition in cellulase production, which was 10–20 folds of that achieved with lactose as inducer, making the crude enzyme more suitable for hydrolyzing pretreated biomass without a necessity for concentration. Moreover, cellulase productivity was consequently increased to 627.1 FPU/L/h, at least 3–5 fold higher than that achieved in cellulase production using soluble inducer lactose and insoluble inducer cellulose as well, saving energy consumption and capital investment significantly for cellulase production by T. reesei. http://dx.doi.org/10.1016/j.nbt.2016.06.959
Abstracts / New Biotechnology 33S (2016) S1–S213
cassette. Since a crucial step for cell wall localization of the enzymes is the secretion of proteins in yeast cells, we also demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the SED1 gene for cell-surface display of cellulolytic enzymes. http://dx.doi.org/10.1016/j.nbt.2016.06.956
S1-2 Designer cellulosome for application to advanced biotechnology tools Sung Ok Han ∗ , Jeong Eun Hyeon, Seung Kyou You Korea University, Republic of Korea The cellulosome is one of nature’s most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. First, to construct the self-assembled biosensor with signal amplification, a cellulosome system, comprising Type I and Type II dockerin–cohesin interactions with different specificity, from the anaerobic Clostridia bacterium was applied. The self-assembled biosensor was highly sensitive and achieved 128.1fold increase in detection levels compared to the control. Second, the designer enzymes and microbes are a key biological technology that can be used for biorefinery. Prior to fermentative production of valuable product from the biomass by microbes, the polymeric substrates are hydrolyzed to release monomeric sugars. The assembly of minicellulosomes by Saccharomyces cerevisiae and Corynebacterium glutamicum increased the activity against various lignocellulosic materials by approximately 3-fold compared with control. Also, red algae-degrading complexes was produced by Escherichia coli and increased the activity against the marine biomass substrate by approximately 2-fold, compared with that for the corresponding enzymes alone. Final, enzyme complex for C1 gas conversion was designed. Carbon monoxide (CO) was successfully efficiently converted by functional complexes containing carbon monoxide dehydrogenase (CODH) and carbon monoxide sensing heme protein (CooA) with enhanced CO binding affinity. An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of CO2 -utilizing Ralstonia eutropha and successfully showed 3.3-fold increased conversion efficiency. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. http://dx.doi.org/10.1016/j.nbt.2016.06.957
S1-3 Hybrid library
nanocellulosome-chimera
designed
from
module
Mitsuo Umetsu Tohoku University, Japan Structural and functional information of proteins are stored by the unit of domain (module) which can be expressed in E. coli, and molecular evolutional techniques prefer to functionalize proteins by the units. Further, in the field of materials, “Nanotechnology” came in use from 1980s, so that various organic and inorganic materials are downsized to nano-meter which is comparable to the size
of the protein domain and modules. In the “smart nano-bio design” we propose, the protein module and various organic/inorganic nanomaterials are considered as “functional nanomaterials” with no distinction of matters, and they are constructively built up in vitro on a scaffold structure of organic or inorganic nanomaterial without being bound to the limitation of structural design using amino acid sequence. In the environmental field, we focus on the enzyme of cellulase. Cellulases are generally modular proteins with independent catalytic and cellulose-binding domains (CBDs), and in some bacteria, catalytic domains (CDs) are non-covalently assembled on a scaffold protein with CBD to form a giant protein complex, called a cellulosome, which efficiently degrades water-insoluble hard materials. Recently, we proposed a new design for artificial cellulosomes with multiple CBDs on non-cellulosome-derived scaffold structures. CDs and CBDs are independently prepared by recombinant means, and they are heterogeneously clustered on inorganic nanoparticle. The heteroclustering of CD with CBD on nanomaterials resulted in significant improvements in the enzyme’s degradation activity for water-insoluble substrates. Here, we show the potential of the combination of nanomaterials and protein module for designing the cellulase with high performance. http://dx.doi.org/10.1016/j.nbt.2016.06.958
S1-4 Overproduction of cellulase by Trichoderma reesei Rut C30 through batch-feeding of synthesized mixture as substrate and inducer Feng-Wu Bai Shanghai Jiao Tong University, China Cellulase for hydrolyzing cellulose in lignocellulosic biomass to release glucose is a prerequisite for producing biofuels and biobased chemicals through microbial fermentation (biorefinery), but high cost with cellulase presents one of the biggest challenges. Here we report the synthesis of low-cost mixture from glucose by -glucosidase through the transglycosylation reaction as substrate for the growth of Trichoderma reesei Rut C30 and inducer for cellulase production. Compared to commonly used soluble inducer lactose, the sugar mixture facilitated mycelial growth, and induced cellulase production more effectively due to the presence of easily assimilated glucose as carbon source for mycelial growth and betadisaccharides, in particular sophorose, for cellulase induction. As a result, cellulase activity as high as 90.3 FPU (filter paper unit)/mL was achieved at 144 h with the fed-batch strategy through which glucose was controlled between 0.05 g/L and 0.30 g/L to eliminate its inhibition in cellulase production, which was 10–20 folds of that achieved with lactose as inducer, making the crude enzyme more suitable for hydrolyzing pretreated biomass without a necessity for concentration. Moreover, cellulase productivity was consequently increased to 627.1 FPU/L/h, at least 3–5 fold higher than that achieved in cellulase production using soluble inducer lactose and insoluble inducer cellulose as well, saving energy consumption and capital investment significantly for cellulase production by T. reesei. http://dx.doi.org/10.1016/j.nbt.2016.06.959