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Production of 3-hydroxypropionic acid through malonyl CoA pathway from glucose by recombinant Escherichia coli BL21
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[B.36] Production of 3-hydroxypropionic acid through malonyl CoA pathway from glucose by recombinant Escherichia coli BL21 Chelladurai Rathnasingh 1 , J. Chrity Catherine 1 , S. Mohan Raj 1,2 , Lee You-Jin 1 , Park Sunghoon 1,∗ 1
Pusan National University, Democratic People’s Republic of Korea Kalasalingam University, India Keywords: 3-Hydroxypropionic acid; Malonyl CoA reductase; Chloroflexus aurantiacus; Acetyl CoA carboxylase 2
3-Hydroxypropionic acid (3-HP) is an important C-3 platform chemical from which several commodity and specialty chemicals can be generated. Previously, we have reported the production of 3HP from glycerol with high titer using recombinant Escherichia coli SH-BGK1 which expresses an adenosylcobalamin-dependent glycerol dehydratase and some active dehydrogenases. However, due to high cost for external addition of vitamin B-12 which is converted to adenosylcobalamin, the process was not economically feasible. In the present study, we studied a new, adenosylcobalamin-free pathway which utilizes malonyl CoA as an immediate substrate of 3-HP. In the new pathway, malonyl CoA is produced from acetyl CoA by acetyl CoA carboxylase in fatty acid metabolic pathway and converted to 3-HP by malonyl CoA reductase (Mcr). The important target enzyme, Mcr, was acquired from Chloroflexus aurantiacus and several recombinant E. coli strains were developed to evaluate the possibility of the new 3-HP production pathway. One recombinant E. coli strain, where only mcr gene of C. aurantiacus was coned and overexpressed, did not produce 3-HP. In other recombinant strains, the genes encoding two more enzymes, acetyl CoA carboxylase (accADBC) and biotinilase (birA), were additionally cloned from E. coli K12 to increase the accumulation of malonyl CoA from acetyl CoA. Furthermore, to accelerate the conversion of NADH to NADPH which is required for the activity of Mcr, transhydrogenase (pntAB) genes of E. coli K12 were overexpressed. The final construct, recombinant E. coli BL21 containing mcr, accADBC, birA and pntAB, showed the production of 1.2 mmol l−1 3-HP from glucose in 24 h under aerobic shake-flask cultivation. The production of 3-HP through malonyl CoA pathway in recombinant E. coli is reported here for the first time. This pathway can contribute to the development of adenosylcobalaminfree process for the production of 3-HP from renewable resources.
doi:10.1016/j.jbiotec.2010.08.070 [B.38] Steady-state PSII direct contribution improves H2 production rates in a C. reinhardtii D1 protein mutant A. Scoma 1,2,∗ , L. Giannelli 2 , G. Torzillo 2 1 Department of Applied Chemistry and Material Science (DICASM), Faculty of Engineering, University of Bologna, Italy 2 Institute for the Study of the Ecosystems (ISE), National Council of Research (CNR), Italy Keywords: Hydrogen; Chlamydomonas; D1 protein; Biophotolysis
doi:10.1016/j.jbiotec.2010.08.069 [B.37] Biological electrochemical devices for production of current and hydrogen A.C. Fisher 1,4,∗ , C. Howe 2,4 , A. Cameron 1,4 , D. Bendall 1,4
tional fossil fuels are considered to be the main contributor to the greenhouse effect; they are subject to a large political risk and destined to unavoidable depletion. Conversely, solar energy is virtually carbon-free, extremely abundant and available worldwide. Nature has clearly demonstrated that it is possible to harness solar energy through the process of photosynthesis. It is estimated that the Earth’s photosynthetic organisms convert over 10 times more energy per year than current human energy consumption, albeit with a low energy conversion efficiency (ca. 0.25%). A number or synthetic techniques have also been developed to try and emulate the photosynthetic process; the most successful of these is the traditional solar cells based on the photovoltaic effect. Unlike photosynthetic organisms they are able to convert energy with a high efficiency (ca. 10%). However, the technology is based on the use of expensive, high purity semi-conductor materials. In order to exploit the advantages of both the biological and synthetic approaches a technology is required which makes use of the high energy conversion efficiency of the synthetic systems whilst keeping the inherent merits of a low-cost biological approach. The Biological Photo-Voltaic (BPV) project aims to develop a novel method for harnessing solar energy which combines the synthetic and biological techniques to produce economical devices with low manufacturing costs, excellent energy conversion efficiency that is virtually carbon-free. The BPV technology is a feasible bio electrochemical platform for generating current, hydrogen, carbon dioxide sequestration, water desalination and co-generation of other by-product. BPV is a multi-disciplinary research effort to understand and develop the solar device by exploiting a wide range of techniques such as: electrochemistry, microfabrication, chemical synthesis, molecular biology, proteomics and numerical simulation.
Smith 3,4 , P.
Bombelli 1,4 , P.
1 Department of Chemical Engineering and Biotechnology, University of Cambridge, United Kingdom 2 Department of Biochemistry, University of Cambridge, United Kingdom 3 Department of Plant sciences, University of Cambridge, United Kingdom 4 Department of Chemistry, University of Bath, United Kingdom Keywords: Photosystem; Fuel cell; Electrochemical
The Sun, in the form of solar energy, is the ultimate source of energy for life on Earth and harnessing this energy is one of the great scientific and technological challenges. Tradi-
Molecular hydrogen (H2 ) has been indicated as one of the most suitable vectors for renewable energies. Among several biological systems, the H2 photoproduction carried out by the green microalga Chlamydomonas reinhardtii has attracted many interests since Melis discovered (2000) that anaerobiosis in the light could be reached by means of sulfur starvation. As a consequence of this, electrons coming from (1) the residual direct photosystem 2 (PSII) activity and (2) endogenous substrates fermentation are photosynthetically driven to a hydrogenase for the release of H2 gas. In this study, the H2 production in a D1 protein mutant of C. reinhardtii (L159I-N230Y) was tested. The physiological capability to evolve high H2 gas amounts showed by this mutant was previously fully described (Torzillo et al., 2009; Scoma, 2010). A standard Rouxlike PBR was equipped with a multiple impeller stirring device developed by us which enhanced photosynthetic efficiency and respiratory capability of cultures (Giannelli et al., 2009; Scoma, 2010). According to the main features of the mutant, culture condi-
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[B.36] Production of 3-hydroxypropionic acid through malonyl CoA pathway from glucose by recombinant Escherichia coli BL21 Chelladurai Rathnasingh 1 , J. Chrity Catherine 1 , S. Mohan Raj 1,2 , Lee You-Jin 1 , Park Sunghoon 1,∗ 1
Pusan National University, Democratic People’s Republic of Korea Kalasalingam University, India Keywords: 3-Hydroxypropionic acid; Malonyl CoA reductase; Chloroflexus aurantiacus; Acetyl CoA carboxylase 2
3-Hydroxypropionic acid (3-HP) is an important C-3 platform chemical from which several commodity and specialty chemicals can be generated. Previously, we have reported the production of 3HP from glycerol with high titer using recombinant Escherichia coli SH-BGK1 which expresses an adenosylcobalamin-dependent glycerol dehydratase and some active dehydrogenases. However, due to high cost for external addition of vitamin B-12 which is converted to adenosylcobalamin, the process was not economically feasible. In the present study, we studied a new, adenosylcobalamin-free pathway which utilizes malonyl CoA as an immediate substrate of 3-HP. In the new pathway, malonyl CoA is produced from acetyl CoA by acetyl CoA carboxylase in fatty acid metabolic pathway and converted to 3-HP by malonyl CoA reductase (Mcr). The important target enzyme, Mcr, was acquired from Chloroflexus aurantiacus and several recombinant E. coli strains were developed to evaluate the possibility of the new 3-HP production pathway. One recombinant E. coli strain, where only mcr gene of C. aurantiacus was coned and overexpressed, did not produce 3-HP. In other recombinant strains, the genes encoding two more enzymes, acetyl CoA carboxylase (accADBC) and biotinilase (birA), were additionally cloned from E. coli K12 to increase the accumulation of malonyl CoA from acetyl CoA. Furthermore, to accelerate the conversion of NADH to NADPH which is required for the activity of Mcr, transhydrogenase (pntAB) genes of E. coli K12 were overexpressed. The final construct, recombinant E. coli BL21 containing mcr, accADBC, birA and pntAB, showed the production of 1.2 mmol l−1 3-HP from glucose in 24 h under aerobic shake-flask cultivation. The production of 3-HP through malonyl CoA pathway in recombinant E. coli is reported here for the first time. This pathway can contribute to the development of adenosylcobalaminfree process for the production of 3-HP from renewable resources.
doi:10.1016/j.jbiotec.2010.08.070 [B.38] Steady-state PSII direct contribution improves H2 production rates in a C. reinhardtii D1 protein mutant A. Scoma 1,2,∗ , L. Giannelli 2 , G. Torzillo 2 1 Department of Applied Chemistry and Material Science (DICASM), Faculty of Engineering, University of Bologna, Italy 2 Institute for the Study of the Ecosystems (ISE), National Council of Research (CNR), Italy Keywords: Hydrogen; Chlamydomonas; D1 protein; Biophotolysis
doi:10.1016/j.jbiotec.2010.08.069 [B.37] Biological electrochemical devices for production of current and hydrogen A.C. Fisher 1,4,∗ , C. Howe 2,4 , A. Cameron 1,4 , D. Bendall 1,4
tional fossil fuels are considered to be the main contributor to the greenhouse effect; they are subject to a large political risk and destined to unavoidable depletion. Conversely, solar energy is virtually carbon-free, extremely abundant and available worldwide. Nature has clearly demonstrated that it is possible to harness solar energy through the process of photosynthesis. It is estimated that the Earth’s photosynthetic organisms convert over 10 times more energy per year than current human energy consumption, albeit with a low energy conversion efficiency (ca. 0.25%). A number or synthetic techniques have also been developed to try and emulate the photosynthetic process; the most successful of these is the traditional solar cells based on the photovoltaic effect. Unlike photosynthetic organisms they are able to convert energy with a high efficiency (ca. 10%). However, the technology is based on the use of expensive, high purity semi-conductor materials. In order to exploit the advantages of both the biological and synthetic approaches a technology is required which makes use of the high energy conversion efficiency of the synthetic systems whilst keeping the inherent merits of a low-cost biological approach. The Biological Photo-Voltaic (BPV) project aims to develop a novel method for harnessing solar energy which combines the synthetic and biological techniques to produce economical devices with low manufacturing costs, excellent energy conversion efficiency that is virtually carbon-free. The BPV technology is a feasible bio electrochemical platform for generating current, hydrogen, carbon dioxide sequestration, water desalination and co-generation of other by-product. BPV is a multi-disciplinary research effort to understand and develop the solar device by exploiting a wide range of techniques such as: electrochemistry, microfabrication, chemical synthesis, molecular biology, proteomics and numerical simulation.
Smith 3,4 , P.
Bombelli 1,4 , P.
1 Department of Chemical Engineering and Biotechnology, University of Cambridge, United Kingdom 2 Department of Biochemistry, University of Cambridge, United Kingdom 3 Department of Plant sciences, University of Cambridge, United Kingdom 4 Department of Chemistry, University of Bath, United Kingdom Keywords: Photosystem; Fuel cell; Electrochemical
The Sun, in the form of solar energy, is the ultimate source of energy for life on Earth and harnessing this energy is one of the great scientific and technological challenges. Tradi-
Molecular hydrogen (H2 ) has been indicated as one of the most suitable vectors for renewable energies. Among several biological systems, the H2 photoproduction carried out by the green microalga Chlamydomonas reinhardtii has attracted many interests since Melis discovered (2000) that anaerobiosis in the light could be reached by means of sulfur starvation. As a consequence of this, electrons coming from (1) the residual direct photosystem 2 (PSII) activity and (2) endogenous substrates fermentation are photosynthetically driven to a hydrogenase for the release of H2 gas. In this study, the H2 production in a D1 protein mutant of C. reinhardtii (L159I-N230Y) was tested. The physiological capability to evolve high H2 gas amounts showed by this mutant was previously fully described (Torzillo et al., 2009; Scoma, 2010). A standard Rouxlike PBR was equipped with a multiple impeller stirring device developed by us which enhanced photosynthetic efficiency and respiratory capability of cultures (Giannelli et al., 2009; Scoma, 2010). According to the main features of the mutant, culture condi-