- Email: [email protected]
Advances in directed evolution of genetic circuits
Abstracts / Journal of Biotechnology 136S (2008) S22–S71 Kaiser, E.T., Lawrence, D.S., 1984. Chemical mutation of enzyme active sites. Science 226, 505–508.
doi:10.1016/j.jbiotec.2008.07.081
S41
I1-P-048 Nitric oxide can enhance puerarin and daidzein accumulation in Pueraria lobata (willd.) ohwi callus under NA+ stress Hu Su 1 , Hu Jiang 2
I1-P-047
1
Process dynamic modeling based on metabolic network of glycerol bioconversion to 1,3-propanediol
Department of Life Science, Jiang Xi Science and Technology Normal University, Nan Chang 330013, China 2 Jiang Xi-OAI, Nan Chang University, Nan Chang 330047, China
Qingrui Zhang 1,2 , Hu Teng 1 , Zhenning Wu 1 , Zhilong Xiu 1,∗
E-mail address: [email protected] (H. Jiang).
1
Department of Biotechnology, Dalian University of Technology, Linggong Road 2, Dalian 116023, China 2 College of Chemical Engineering, Qingdao University of Science and Technology, Zhengzhou Road 53, Qingdao 266042, China E-mail address: [email protected] (Z. Xiu).
1,3-Propanediol is considered to be one of the bulk chemicals, which is likely to be produced by bioprocesses on large scales. The issue of bioprocess modeling from extracellular measurements has been considered for a long time. The traditional macroscopic models of glycerol fermentation are all based on unstructured models in which the cells are just viewed as a catalyst for the conversion of substrates into products without paying much attention to the intracellular behaviour (Xiu et al., 2004). Last decades, the constraint-based metabolic network models were developed and were used for analyzing the intracellular flux distribution (Edwards and Palsson, 2000). However, the steady-state constraintbased models are essentially stoichiometric models, still containing no information regarding reaction kinetics. Recently, some published studies demonstrated using the metabolic networks for the dynamic metabolic modeling (Goudar et al., 2006). However, no attempt has been made on the glycerol metabolic system. In this work, a macro-element model incorporating intracellular metabolism was developed. Firstly, the available structure of the metabolic network is determined by using the method of metabolic flux analysis (MFA) through which to check the consistency of the assumed metabolic network with the experimental data. Then the metabolic network was decomposed into elementary modes, which are the simplest paths able to operate coherently in steady-state. A reduced reaction mechanism in the form of simplified reactions connecting substrates with end-products was obtained. Finally, the metabolic dynamic model was established according to the elementary modes and was employed to analyze experimental data of glycerol fermentation under anaerobic condition. The prediction of the time evolution was in good agreement with the experimental data. In addition, the error analysis revealed that the glycerol consumption and 1,3-PD formation were coupling with the cell growth but the ethanol formation was not. References Edwards, J.S., Palsson, B.O., 2000. The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. Proc. Natl. Acad. Sci. U.S.A. 97, 5528–5533. Goudar, C., Biener, R., Zhang, C., Michaels, J., Piret, J., Konstantinov, K., 2006. Towards industrial application of quasi real-time metabolic flux analysis for mammalian cell culture. Adv. Biochem. Eng. Biotechnol. 101, 99–118. Xiu, Z.L., Song, B.H., Wang, Z.T., Sun, L.H., Feng, E.M., Zeng, A.P., 2004. Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in oneand two-stage anaerobic cultures. Biochem. Eng. J. 19, 189–197.
doi:10.1016/j.jbiotec.2008.07.082
In order to study the nitric oxide function (Clarke et al., 2000) in puerarin and daidzein accumulation under Na+ stress in Pueraria lobata (willd.) ohwi callus, NaCl (NaCl plus SNP, NaCl/SNP plus CPTIO, etc.) was added to suspension callus (Abdul and Alia, 2006). The concentration of puerarin and daidzein in the callus was tested by HPLC. The result indicated that 150 mM Na+ can induce puerarin and daidzein accumulation, the accumulation level can be enhanced by NO (nitric oxide) donated by SNP (Maojun et al., 2006), puerarin concentration increased from 0.0766 to 0.2166 mg g−1 (dry weight) within 51 h, daidzein concentration was lower under Na+ stress within the initial 24 h, but increased to a higher level (0.0117–0.0181 mg g−1 ). CPTIO as the specific scavenger of nitric oxide induced higher-level accumulation of puerarin (max 0.2946 mg g−1 ) and daidzein (max 0.038 mg g−1 ) instead of inhibiting at the early stage. Acknowledgement The authors thank Prof. Maojun Xu in Zhejiang Gongshang University for providing the Pueraria lobata (willd.) ohwi callus. References Abdul, Wahid, Alia, Ghazanfar, 2006. Possible involvement of some secondary metabolites in salt tolerance of sugarcane. Plant Physiol. 163, 723–730. Clarke, A., Desikan, R., Hurst, R.D., Hancock, J.T., Neill, S.J., 2000. NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures. Plant J. 24, 667–677. Maojun, Xu, Jufang, Dong, Muyaun, Zhu, 2006. Nitric oxide mediates the fungal elicitor-induced puerarin production in Pueraria thomsonii Benth suspension cells through a salicylic acid (SA)-dependent or a jasmonic acid (JA)-dependent signal pathway. Science in China C: Life Science 36, 66–75.
doi:10.1016/j.jbiotec.2008.07.083 I1-P-049 Advances in directed evolution of genetic circuits Junyang Yue ∗ , Jincong Yu, Baishan Fang Key Laboratory of Industrial Biotechnology (Huaqiao University), Fujian Province University, Quanzhou 362021, PR China E-mail address: [email protected] (B. Fang). Since the completion of the sequencing of large number genomes, especially with the end of the Human Genome Project, the term “functional genomics” has arisen, which refers to the attempts to apply large-scale strategies to analyze gene function and protein function. Papers are increasingly being reported to gene-functionoriented, including protein–DNA interactions, protein–protein interactions, messenger RNA stabilities, protein stabilities, and interactions between protein, DNA, RNA, and small molecules. These help us to gain insight into how natural genetic networks function with remarkable robustness, stability, and adaptability to the complicated and sophisticated biological systems and changing environments. By studying and simulating such networks, biolog-
S42
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
ical genetic circuit engineers could design and synthesize a set of simple genetic circuits, which are artificial networks of transcriptional control elements inserted into living cells in order to “program” cellular behavior. Furthermore, engineers can extend this application to predicting and programming biological behavior and performance inside living cells. Directed evolution is a momentous and significant approach we believe to be generally applicable for studying and optimizing targeted circuits even for which are not being sufficient well-known to engineer by conventional “rational” approaches. Here, we give a review how directed evolution has achieved to assist the engineering and building of genetic circuits and artificial cells. Directed evolution is already being successfully used for the creation of both RNA and protein and of not only unicellular organism but also multicellular organism; and recently being remarkably applied with the domination of artificial networks to surmount circuit components. References Timothy, S., Gardner, Charles, R., Cantor, James, J., Collins, 2000. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342. Yohei, Yokobayashi, Ron, Weiss, Frances, H. Arnold, 2002. Directed evolution of a genetic circuit. Proceedings of the National Academy of Sciences 99, 16587–16591. Yohei, Yokobayashi, Cynthia, H. Collins, Jared, R. Leadbetter, Ron, Weiss, Frances, H. Arnold, 2003. Evolutionary design of genetic circuits and cell–cell communications. Advances in Complex Systems 6, 1–9.
doi:10.1016/j.jbiotec.2008.07.084 I1-P-050 T-muurolol sesquiterpenes from marine Streptomyces sp. M491 and revision of the configurations for the previously reported sesquiterpenes Ling Ding 1,2,∗ , Song Qin 1 , Fuchao Li 1 , Hartmut Laatsch 2 1
The Institute of Oceanology, Chinese Academy of Sciences, Nanhai Rd. 7, Qingdao 266071, China 2 Department of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany E-mail address: [email protected] (L. Ding). In the previous investigation of metabolites from the marine Streptomyces sp. M491 isolated from the Qingdao coast (China), we reported three new sesquiterpenes (Wu et al., 2007). The continuing research on this strain led to further isolation of another four sesquiterpenes. 15-Hydroxy-T-muurolol and 11,15dihydroxy-T-muurolol along with known compounds T-muurolol and 3␣-hydroxy-T-muurolol which are first reported here as microbial metabolites. 3-Oxo-T-muurolol was re-isolated and the crystal structure was obtained. The absolute configuration of all the other sesquiterpenes was determined through NOESY analysis and comparison with 3-oxo-T-muurolol. This led to a revision of the previously reported configuration (Wu et al., 2007). The sesquiterpenes have been tested for their cytotoxicity against 37 human tumor cell lines using MTT method. Except 15-hydroxy-T-muurolol which showed weak activity (average IC50 = 6.7 g/mL), the others were not cytotoxic. Reference Wu, S.J., Fotso, S., Li, F., Qin, S., Laatsch, H., 2007. Amorphane sesquiterpenes from a marine Streptomyces sp. J. Nat. Prod. 70, 304–306.
doi:10.1016/j.jbiotec.2008.07.085
I1-P-051 The effects of different light treatments and gibberellin on establishment of parasitism between dodder and its host Dongxiao Li 1,2,∗ , Huihuang Zhang 2 , Jingling Chang 1 , Liang Chen 2 1 2
Henan Institute of Science of Technology, Xinxiang 453003, China School of Life Sciences, Xiamen University, Xiamen 361005, China
E-mail address: [email protected] (D. Li). Dodders are holoparasitic higher plants that can invade a large range of species, which indicates they must have highly adaptable mechanisms for host attachment. Unfortunately, the nature of the mechanism(s) is still unknown. The occurrence of twining and haustoria formation is regarded as the sign of successful parasite and thus became the focus of studies on host-parasite interactions. Furthermore, little is known about the effect of GAs on these processes, which regulate many aspects of plant development. In this study, we have investigated the effects of different light treatments on hook opening and twining of Cuscuta australis and establishment of parasitism between dodders and Medicago sativa or plastic rods with LEDs as light sources for the first time. The effects of GA3 and GAs biosynthesis inhibitor paclobutrazol on these responses have also been investigated. Parasitism was observed between C. australis and Medicago sativa in sunlight or under blue light, but no parasitism occurred in darkness, under white or red light. It suggested light is a necessary factor for dodders to parasite the hosts successfully and even if other factors were satisfied, dodders still did not wrap around a potential host if no appropriate light existed. Moreover, dodders displayed a higher percentage of twining in shorter durations of light illumination between dodder and M. sativa comparing with that between dodder and plastic rods. It suggested chemical signals might facilitate host recognition and twining response. Blue light displayed different roles for controlling hook opening and twining in a continuous process which were proved to be controlled by Pfr and Pr, respectively (Lane and Kasperbauer, 1965; Tada et al., 1996; Haidar, 2003) and it suggested there may exist an intrinsic modulating mechanism to regulate transition of these roles. The negative effect of paclobutrazol on twining response was reversed by addition of GA3 and it suggested GA3 is involved in controlling this response. References Haidar, M.A., 2003. Characterisation of the interaction between cryptochromes and phytochromes in blue light-induced coiling and prehaustoria development of dodder (Cuscuta campestris) seedlings. Ann. Appl. Biol. 143, 57–62. Lane, H.C., Kasperbauer, M.J., 1965. Photomorphogenic responses of dodder seedlings. Plant Physiol. 40, 109–116. Tada, Y., Sugai, M., Furuhashi, K., 1996. Haustoria of Cuscuta japonica, a holoparasitic flowering plant, are induced by the cooperative effects of far-red light and tactile stimuli. Plant Cell Physiol. 37, 1049–1053.
doi:10.1016/j.jbiotec.2008.07.086
doi:10.1016/j.jbiotec.2008.07.081
S41
I1-P-048 Nitric oxide can enhance puerarin and daidzein accumulation in Pueraria lobata (willd.) ohwi callus under NA+ stress Hu Su 1 , Hu Jiang 2
I1-P-047
1
Process dynamic modeling based on metabolic network of glycerol bioconversion to 1,3-propanediol
Department of Life Science, Jiang Xi Science and Technology Normal University, Nan Chang 330013, China 2 Jiang Xi-OAI, Nan Chang University, Nan Chang 330047, China
Qingrui Zhang 1,2 , Hu Teng 1 , Zhenning Wu 1 , Zhilong Xiu 1,∗
E-mail address: [email protected] (H. Jiang).
1
Department of Biotechnology, Dalian University of Technology, Linggong Road 2, Dalian 116023, China 2 College of Chemical Engineering, Qingdao University of Science and Technology, Zhengzhou Road 53, Qingdao 266042, China E-mail address: [email protected] (Z. Xiu).
1,3-Propanediol is considered to be one of the bulk chemicals, which is likely to be produced by bioprocesses on large scales. The issue of bioprocess modeling from extracellular measurements has been considered for a long time. The traditional macroscopic models of glycerol fermentation are all based on unstructured models in which the cells are just viewed as a catalyst for the conversion of substrates into products without paying much attention to the intracellular behaviour (Xiu et al., 2004). Last decades, the constraint-based metabolic network models were developed and were used for analyzing the intracellular flux distribution (Edwards and Palsson, 2000). However, the steady-state constraintbased models are essentially stoichiometric models, still containing no information regarding reaction kinetics. Recently, some published studies demonstrated using the metabolic networks for the dynamic metabolic modeling (Goudar et al., 2006). However, no attempt has been made on the glycerol metabolic system. In this work, a macro-element model incorporating intracellular metabolism was developed. Firstly, the available structure of the metabolic network is determined by using the method of metabolic flux analysis (MFA) through which to check the consistency of the assumed metabolic network with the experimental data. Then the metabolic network was decomposed into elementary modes, which are the simplest paths able to operate coherently in steady-state. A reduced reaction mechanism in the form of simplified reactions connecting substrates with end-products was obtained. Finally, the metabolic dynamic model was established according to the elementary modes and was employed to analyze experimental data of glycerol fermentation under anaerobic condition. The prediction of the time evolution was in good agreement with the experimental data. In addition, the error analysis revealed that the glycerol consumption and 1,3-PD formation were coupling with the cell growth but the ethanol formation was not. References Edwards, J.S., Palsson, B.O., 2000. The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. Proc. Natl. Acad. Sci. U.S.A. 97, 5528–5533. Goudar, C., Biener, R., Zhang, C., Michaels, J., Piret, J., Konstantinov, K., 2006. Towards industrial application of quasi real-time metabolic flux analysis for mammalian cell culture. Adv. Biochem. Eng. Biotechnol. 101, 99–118. Xiu, Z.L., Song, B.H., Wang, Z.T., Sun, L.H., Feng, E.M., Zeng, A.P., 2004. Optimization of dissimilation of glycerol to 1,3-propanediol by Klebsiella pneumoniae in oneand two-stage anaerobic cultures. Biochem. Eng. J. 19, 189–197.
doi:10.1016/j.jbiotec.2008.07.082
In order to study the nitric oxide function (Clarke et al., 2000) in puerarin and daidzein accumulation under Na+ stress in Pueraria lobata (willd.) ohwi callus, NaCl (NaCl plus SNP, NaCl/SNP plus CPTIO, etc.) was added to suspension callus (Abdul and Alia, 2006). The concentration of puerarin and daidzein in the callus was tested by HPLC. The result indicated that 150 mM Na+ can induce puerarin and daidzein accumulation, the accumulation level can be enhanced by NO (nitric oxide) donated by SNP (Maojun et al., 2006), puerarin concentration increased from 0.0766 to 0.2166 mg g−1 (dry weight) within 51 h, daidzein concentration was lower under Na+ stress within the initial 24 h, but increased to a higher level (0.0117–0.0181 mg g−1 ). CPTIO as the specific scavenger of nitric oxide induced higher-level accumulation of puerarin (max 0.2946 mg g−1 ) and daidzein (max 0.038 mg g−1 ) instead of inhibiting at the early stage. Acknowledgement The authors thank Prof. Maojun Xu in Zhejiang Gongshang University for providing the Pueraria lobata (willd.) ohwi callus. References Abdul, Wahid, Alia, Ghazanfar, 2006. Possible involvement of some secondary metabolites in salt tolerance of sugarcane. Plant Physiol. 163, 723–730. Clarke, A., Desikan, R., Hurst, R.D., Hancock, J.T., Neill, S.J., 2000. NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures. Plant J. 24, 667–677. Maojun, Xu, Jufang, Dong, Muyaun, Zhu, 2006. Nitric oxide mediates the fungal elicitor-induced puerarin production in Pueraria thomsonii Benth suspension cells through a salicylic acid (SA)-dependent or a jasmonic acid (JA)-dependent signal pathway. Science in China C: Life Science 36, 66–75.
doi:10.1016/j.jbiotec.2008.07.083 I1-P-049 Advances in directed evolution of genetic circuits Junyang Yue ∗ , Jincong Yu, Baishan Fang Key Laboratory of Industrial Biotechnology (Huaqiao University), Fujian Province University, Quanzhou 362021, PR China E-mail address: [email protected] (B. Fang). Since the completion of the sequencing of large number genomes, especially with the end of the Human Genome Project, the term “functional genomics” has arisen, which refers to the attempts to apply large-scale strategies to analyze gene function and protein function. Papers are increasingly being reported to gene-functionoriented, including protein–DNA interactions, protein–protein interactions, messenger RNA stabilities, protein stabilities, and interactions between protein, DNA, RNA, and small molecules. These help us to gain insight into how natural genetic networks function with remarkable robustness, stability, and adaptability to the complicated and sophisticated biological systems and changing environments. By studying and simulating such networks, biolog-
S42
Abstracts / Journal of Biotechnology 136S (2008) S22–S71
ical genetic circuit engineers could design and synthesize a set of simple genetic circuits, which are artificial networks of transcriptional control elements inserted into living cells in order to “program” cellular behavior. Furthermore, engineers can extend this application to predicting and programming biological behavior and performance inside living cells. Directed evolution is a momentous and significant approach we believe to be generally applicable for studying and optimizing targeted circuits even for which are not being sufficient well-known to engineer by conventional “rational” approaches. Here, we give a review how directed evolution has achieved to assist the engineering and building of genetic circuits and artificial cells. Directed evolution is already being successfully used for the creation of both RNA and protein and of not only unicellular organism but also multicellular organism; and recently being remarkably applied with the domination of artificial networks to surmount circuit components. References Timothy, S., Gardner, Charles, R., Cantor, James, J., Collins, 2000. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342. Yohei, Yokobayashi, Ron, Weiss, Frances, H. Arnold, 2002. Directed evolution of a genetic circuit. Proceedings of the National Academy of Sciences 99, 16587–16591. Yohei, Yokobayashi, Cynthia, H. Collins, Jared, R. Leadbetter, Ron, Weiss, Frances, H. Arnold, 2003. Evolutionary design of genetic circuits and cell–cell communications. Advances in Complex Systems 6, 1–9.
doi:10.1016/j.jbiotec.2008.07.084 I1-P-050 T-muurolol sesquiterpenes from marine Streptomyces sp. M491 and revision of the configurations for the previously reported sesquiterpenes Ling Ding 1,2,∗ , Song Qin 1 , Fuchao Li 1 , Hartmut Laatsch 2 1
The Institute of Oceanology, Chinese Academy of Sciences, Nanhai Rd. 7, Qingdao 266071, China 2 Department of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstrasse 2, D-37077 Göttingen, Germany E-mail address: [email protected] (L. Ding). In the previous investigation of metabolites from the marine Streptomyces sp. M491 isolated from the Qingdao coast (China), we reported three new sesquiterpenes (Wu et al., 2007). The continuing research on this strain led to further isolation of another four sesquiterpenes. 15-Hydroxy-T-muurolol and 11,15dihydroxy-T-muurolol along with known compounds T-muurolol and 3␣-hydroxy-T-muurolol which are first reported here as microbial metabolites. 3-Oxo-T-muurolol was re-isolated and the crystal structure was obtained. The absolute configuration of all the other sesquiterpenes was determined through NOESY analysis and comparison with 3-oxo-T-muurolol. This led to a revision of the previously reported configuration (Wu et al., 2007). The sesquiterpenes have been tested for their cytotoxicity against 37 human tumor cell lines using MTT method. Except 15-hydroxy-T-muurolol which showed weak activity (average IC50 = 6.7 g/mL), the others were not cytotoxic. Reference Wu, S.J., Fotso, S., Li, F., Qin, S., Laatsch, H., 2007. Amorphane sesquiterpenes from a marine Streptomyces sp. J. Nat. Prod. 70, 304–306.
doi:10.1016/j.jbiotec.2008.07.085
I1-P-051 The effects of different light treatments and gibberellin on establishment of parasitism between dodder and its host Dongxiao Li 1,2,∗ , Huihuang Zhang 2 , Jingling Chang 1 , Liang Chen 2 1 2
Henan Institute of Science of Technology, Xinxiang 453003, China School of Life Sciences, Xiamen University, Xiamen 361005, China
E-mail address: [email protected] (D. Li). Dodders are holoparasitic higher plants that can invade a large range of species, which indicates they must have highly adaptable mechanisms for host attachment. Unfortunately, the nature of the mechanism(s) is still unknown. The occurrence of twining and haustoria formation is regarded as the sign of successful parasite and thus became the focus of studies on host-parasite interactions. Furthermore, little is known about the effect of GAs on these processes, which regulate many aspects of plant development. In this study, we have investigated the effects of different light treatments on hook opening and twining of Cuscuta australis and establishment of parasitism between dodders and Medicago sativa or plastic rods with LEDs as light sources for the first time. The effects of GA3 and GAs biosynthesis inhibitor paclobutrazol on these responses have also been investigated. Parasitism was observed between C. australis and Medicago sativa in sunlight or under blue light, but no parasitism occurred in darkness, under white or red light. It suggested light is a necessary factor for dodders to parasite the hosts successfully and even if other factors were satisfied, dodders still did not wrap around a potential host if no appropriate light existed. Moreover, dodders displayed a higher percentage of twining in shorter durations of light illumination between dodder and M. sativa comparing with that between dodder and plastic rods. It suggested chemical signals might facilitate host recognition and twining response. Blue light displayed different roles for controlling hook opening and twining in a continuous process which were proved to be controlled by Pfr and Pr, respectively (Lane and Kasperbauer, 1965; Tada et al., 1996; Haidar, 2003) and it suggested there may exist an intrinsic modulating mechanism to regulate transition of these roles. The negative effect of paclobutrazol on twining response was reversed by addition of GA3 and it suggested GA3 is involved in controlling this response. References Haidar, M.A., 2003. Characterisation of the interaction between cryptochromes and phytochromes in blue light-induced coiling and prehaustoria development of dodder (Cuscuta campestris) seedlings. Ann. Appl. Biol. 143, 57–62. Lane, H.C., Kasperbauer, M.J., 1965. Photomorphogenic responses of dodder seedlings. Plant Physiol. 40, 109–116. Tada, Y., Sugai, M., Furuhashi, K., 1996. Haustoria of Cuscuta japonica, a holoparasitic flowering plant, are induced by the cooperative effects of far-red light and tactile stimuli. Plant Cell Physiol. 37, 1049–1053.
doi:10.1016/j.jbiotec.2008.07.086