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Design and optimization of a novel DNA scaffold system for the construction of artificial enzymatic complexes
New Biotechnology · Volume 29S · September 2012
ples were screened for new lactobacilli isolates using selective (Rogosa) medium. Over 149 isolates were isolated from human vagina and saliva, soil, water, plain yogurt, shellfishes and kimchi. D-Lactic acid production by individual isolates was examined using HPLC analysis with Chiralpak MA column and UV detector. PCR amplified ldhD gene from Lactobacillus rhamnosus ATCC53103 was cloned using a Lactobacillus/E. coli shuttle vector, pTRKH2. pTRKH2::ldhD was constructed using enzyme restriction and ligation, and was introduced into L. plantarum, L. paracasei, and L. gasseri by electroporation for over-expression of D-lactic acid. Transformants were confirmed by colony PCR method and compared with its wild types in growth and pH change. In several transformants, D-lactic acid production was increased up to 50% compared to that of wild types. This study confirms the potential of strain improvement for the production of renewable resources such as D-lactic acid. Further studies include use of 3L fermentor for scale-up study, performance of various stress tests on selected transformants and co-culture of the transformant with Shewanella oneidensis MR-1. Keywords: Lactobacillus; D-Lactic acid; Recombinant strain; pTRKH2; Transformation http://dx.doi.org/10.1016/j.nbt.2012.08.134 Poster 1.2.03 Design and optimization of a novel DNA scaffold system for the construction of artificial enzymatic complexes Jun Hyoung Lee KAIST, Daejeon, South Korea Cellular and metabolic reactions in a living organism are orchestrated as complex networks of individual reactions that seem as though they were precisely designed and ordered for the survival of the living organism. However, the actual reactions are mediated by simple diffusion and random collisions of metabolites and enzymes, which cause metabolic reactions to be inefficient by lowering the local metabolite concentration around the enzymes. Furthermore, the presence of toxic intermediates within a host cell inhibits the activity of many cellular functions. These intrinsic drawbacks of metabolic reactions have prompted researchers to develop novel biological systems that make enzymatic reactions more efficient. Toward this end, we designed a novel DNA scaffold system, in which a zinc finger protein (ZFP) serves as an adapter for the site-specific binding of each metabolic enzyme of interest to a precisely ordered location on a DNA double helix, to increase the proximity of enzymes and the local concentration of metabolites to maximize the production of a substance of interest. Application of our DNA scaffold system to L-threonine production significantly increased the efficiency of the threonine biosynthetic pathway in Escherichia coli, substantially reducing the production time for threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the L-threonine-producing strain by reducing the intracellular concentration of toxic intermediates such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial enzymatic
complexes as well as for the production of many useful biomaterials. http://dx.doi.org/10.1016/j.nbt.2012.08.135 Poster 1.2.04 Focused proteome analysis of cellulosome of Clostridium cellulovorans Kazuma Matsui 1,∗ , Hironobu Morisaka 1 , Kouichi Yutaka Tamaru 2 , Hideo Miyake 2 , Mitsuyoshi Ueda 1
Kuroda 1 ,
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2 Department of Life Science, Mie University Graduate School of Bioresourses, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan Clostridium cellulovorans is an anaerobic and mesophilic bacterium suitable for degradation of cellulosic biomass. The cellulosome is a large extracellular enzyme complex containing a scaffolding protein and a number of cellulosomal enzymes, and contributes to efficient degradation of plant cell wall polysaccharides. Through the interaction between cohesins of a scaffolding protein and dockerins of cellulosomal enzymes, ‘cellulosome’ is built up. Based on whole-genome sequencing of C. cellulovorans, a main scaffolding protein having 9 cohesin domains and 53 cellulosomal enzymes having dockerin domains were found. The diversity in cellulosomal enzymes allows C. cellulovorans to utilize different types of cellulosic materials. In addition, C. cellulovorans varies the composition of cellulosomal enzymes according to the type of substrates. However, the mechanisms for optimizing the combination of cellulosomal enzymes are not clear. Thus, identification and quantitative analyses of the cellulosomal enzymes in the presence of various substrates were attempted. Previously, the cellulosomal proteins of C. cellulovorans were separated by 2D-PAGE and have been identified. However, the number of identified proteins was much lower and only a part of cellulosomal enzymes according to the type of substrates were detected. Therefore, comprehensive proteome analysis focusing on cellulosome of C. cellulovorans was performed. After the isolation of cellulosomal proteins from media, separation of peptides from cellulosomal proteins was carried out using the HPLC system with a 300 cm wide-pore monolithic column. Consequently, the composition and ratio of cellulosomal enzymes according to the type of substrates have been clarified. http://dx.doi.org/10.1016/j.nbt.2012.08.136
www.elsevier.com/locate/nbt S49
ples were screened for new lactobacilli isolates using selective (Rogosa) medium. Over 149 isolates were isolated from human vagina and saliva, soil, water, plain yogurt, shellfishes and kimchi. D-Lactic acid production by individual isolates was examined using HPLC analysis with Chiralpak MA column and UV detector. PCR amplified ldhD gene from Lactobacillus rhamnosus ATCC53103 was cloned using a Lactobacillus/E. coli shuttle vector, pTRKH2. pTRKH2::ldhD was constructed using enzyme restriction and ligation, and was introduced into L. plantarum, L. paracasei, and L. gasseri by electroporation for over-expression of D-lactic acid. Transformants were confirmed by colony PCR method and compared with its wild types in growth and pH change. In several transformants, D-lactic acid production was increased up to 50% compared to that of wild types. This study confirms the potential of strain improvement for the production of renewable resources such as D-lactic acid. Further studies include use of 3L fermentor for scale-up study, performance of various stress tests on selected transformants and co-culture of the transformant with Shewanella oneidensis MR-1. Keywords: Lactobacillus; D-Lactic acid; Recombinant strain; pTRKH2; Transformation http://dx.doi.org/10.1016/j.nbt.2012.08.134 Poster 1.2.03 Design and optimization of a novel DNA scaffold system for the construction of artificial enzymatic complexes Jun Hyoung Lee KAIST, Daejeon, South Korea Cellular and metabolic reactions in a living organism are orchestrated as complex networks of individual reactions that seem as though they were precisely designed and ordered for the survival of the living organism. However, the actual reactions are mediated by simple diffusion and random collisions of metabolites and enzymes, which cause metabolic reactions to be inefficient by lowering the local metabolite concentration around the enzymes. Furthermore, the presence of toxic intermediates within a host cell inhibits the activity of many cellular functions. These intrinsic drawbacks of metabolic reactions have prompted researchers to develop novel biological systems that make enzymatic reactions more efficient. Toward this end, we designed a novel DNA scaffold system, in which a zinc finger protein (ZFP) serves as an adapter for the site-specific binding of each metabolic enzyme of interest to a precisely ordered location on a DNA double helix, to increase the proximity of enzymes and the local concentration of metabolites to maximize the production of a substance of interest. Application of our DNA scaffold system to L-threonine production significantly increased the efficiency of the threonine biosynthetic pathway in Escherichia coli, substantially reducing the production time for threonine (by over 50%). In addition, this DNA scaffold system enhanced the growth rate of the L-threonine-producing strain by reducing the intracellular concentration of toxic intermediates such as homoserine. Our DNA scaffold system can be used as a platform technology for the construction and optimization of artificial enzymatic
complexes as well as for the production of many useful biomaterials. http://dx.doi.org/10.1016/j.nbt.2012.08.135 Poster 1.2.04 Focused proteome analysis of cellulosome of Clostridium cellulovorans Kazuma Matsui 1,∗ , Hironobu Morisaka 1 , Kouichi Yutaka Tamaru 2 , Hideo Miyake 2 , Mitsuyoshi Ueda 1
Kuroda 1 ,
1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 2 Department of Life Science, Mie University Graduate School of Bioresourses, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan Clostridium cellulovorans is an anaerobic and mesophilic bacterium suitable for degradation of cellulosic biomass. The cellulosome is a large extracellular enzyme complex containing a scaffolding protein and a number of cellulosomal enzymes, and contributes to efficient degradation of plant cell wall polysaccharides. Through the interaction between cohesins of a scaffolding protein and dockerins of cellulosomal enzymes, ‘cellulosome’ is built up. Based on whole-genome sequencing of C. cellulovorans, a main scaffolding protein having 9 cohesin domains and 53 cellulosomal enzymes having dockerin domains were found. The diversity in cellulosomal enzymes allows C. cellulovorans to utilize different types of cellulosic materials. In addition, C. cellulovorans varies the composition of cellulosomal enzymes according to the type of substrates. However, the mechanisms for optimizing the combination of cellulosomal enzymes are not clear. Thus, identification and quantitative analyses of the cellulosomal enzymes in the presence of various substrates were attempted. Previously, the cellulosomal proteins of C. cellulovorans were separated by 2D-PAGE and have been identified. However, the number of identified proteins was much lower and only a part of cellulosomal enzymes according to the type of substrates were detected. Therefore, comprehensive proteome analysis focusing on cellulosome of C. cellulovorans was performed. After the isolation of cellulosomal proteins from media, separation of peptides from cellulosomal proteins was carried out using the HPLC system with a 300 cm wide-pore monolithic column. Consequently, the composition and ratio of cellulosomal enzymes according to the type of substrates have been clarified. http://dx.doi.org/10.1016/j.nbt.2012.08.136
www.elsevier.com/locate/nbt S49