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Signal transduction engineering: a powerful platform technology for enhancing secondary metabolite production
New Biotechnology · Volume 31S · July 2014
with fluorescently labelled B12 . The uptake mechanism remains unclear and is part of our current research. An understanding of transport along the protein shell and its selectivity is an important landmark in our efforts to generate engineered compartments capable of sequestering complex synthetic metabolic pathways.
SYMPOSIUM 5: SYNTHETIC BIOLOGY
O5-5 Novel tuneable gene expression systems based on orthogonal riboswitches Neil Dixon MIB – Manchester, United Kingdom
http://dx.doi.org/10.1016/j.nbt.2014.05.1663
O5-4 Use of transporter plug-ins in building effective microbial cell factories for chemical and fuel production Christopher Grant ∗ , Phattaraporn Morris, Frank Baganz University College London, United Kingdom
Synthetic biology hopes to predictably build cellular factories for the production of chemicals, pharmaceuticals and fuels from cheap renewable feedstocks. A critical, yet understudied, element of this is control of compound transport both into and out of the cell as a means of avoiding issues such as substrate access limitations, substrate and product inhibition and aiding product recovery. We report here a successful strategy for the contextual evaluation of this topic using a library of transport modifying plug-ins combined with multifactorial characterisation. Auxiliary plasmids (pUMP) were used to express a library of transporters as plug-ins alongside two biosynthesis plasmids pGEC41, which oxidises hydrocarbons into fatty alcohols and pADAR7942 which synthesizes bioalkanes from metabolic fatty acid precursors. We demonstrate here benefits of the transporter plug-in approach for: (i) Facilitated delivery hydrophobic substrates to improve whole-cell biocatalysis rates by up to 70 fold (ii) Industrially relevant product yields of over 40 g/Lorganic phase (8 g/Ltotal ) (iii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols (iv) Improving bioalkane synthesis yields from glycerol by >5 fold (v) Reducing alkanol and aldehyde intermediate formation in biosynthesis of bioalkanes by > 10 fold (vi) The integration with in situ product removal strategies to improve bioalkane yields by 10 fold compared to the starting process. This library and plug-in approach is of broad appeal for biological production of hydrophobic compounds and could be a key enabling technology for biological routes for producing a wider range of hydrophobic compounds such as biofuels, fine and specialty chemicals and pharmaceutical intermediates. http://dx.doi.org/10.1016/j.nbt.2014.05.1664
Strategies that permit precisely controlled, differential, and simultaneous expression of multiple genes would be extremely useful for a broad range of metabolic engineering, protein expression and synthetic biology applications. A new paradigm in genetic regulation emerged with the discovery of novel genetic regulatory elements within the 5 UTR of bacterial mRNA. Upon binding to a specific metabolite, these so-called ‘riboswitches’ change conformation, permitting differential gene regulation to occur. As these switches operate via a small molecule-dependent, protein-free mechanism, they present themselves as attractive targets for use as novel genetic control elements. Previously, we showed that it is possible to re-engineer riboswitches so that they no longer bind to their original cognate ligands, but are instead activated by synthetic ligands, and further that these ‘orthogonal’ riboswitches can be used to control heterologous gene expression in vivo [Dixon et al., PNAS 2010]. We have further developed these into multi-component systems that permit fine-tuning over co-expression output stoichiometry, with wide ranging potential applications in functional and structural analysis [Dixon et al., Angew Chem, 2012]. Finally, I will discuss the development of these cellular systems and molecular devices from simply proof-of-principle studies, into the tuneable modular expression system RiboTite, and demonstrate the application of this expression technology for the production of proteins of biotechnological interest. http://dx.doi.org/10.1016/j.nbt.2014.05.1665
O5-6 Signal transduction engineering: a powerful platform technology for enhancing secondary metabolite production Jian-Jiang Zhong ∗ , Yi-Ning Xu, Gao-Yi Tan, Linquan Bai Shanghai Jiao Tong University, China
Streptomycetes and higher fungi produce many bioactive secondary metabolites. Ganoderic acids (GAs) produced by Ganoderma lucidum, a higher fungus, have significant anti-tumor and anti-metastasis activities. Validamycin, an anti-fungal antibiotic produced by Streptomyces hygroscopicus 5008, is an efficient rice sheath blight controller, and can be used as the source for chemical synthesis of voglibos, an antidiabetic drug. Engineering of regulatory mechanism was done to enhance the production of target secondary metabolites. The effects of metal ions on the GAs biosynthesis in liquid cultures of G. lucidum were investigated, and the increased enzyme activities and up-regulation of transcriptional levels of genes in the triterpene biosynthesis were www.elsevier.com/locate/nbt S23
SYMPOSIUM 5: SYNTHETIC BIOLOGY
observed. The regulation mechanism of Mn2+ on the GA biosynthesis was found to be via calcineurin signaling transduction. For the validamycin biosynthesis, our previous study indicated the involvement of A-factor-like cascade. A recent genome-wide analysis reveals three pairs of afsA-arpA in S. hygroscopicus 5008. This work aims to decipher the regulatory role of the multiple afsA-arpA homologs in the A-factor-like cascade, and then to improve the validamycin production by engineering the regulatory cascade. By double deletion of shbR1/R3, the transcriptions of adpA-H and the validamycin biosynthetic genes were up-regulated, and the validamycin production and productivity were enhanced significantly for both the wide-type and a high-producing industrial strain. The transcriptomic analysis revealed that the engineering of A-factorlike signaling cascade caused a shift from primary to secondary metabolism. The signal transduction engineering proposed here is very useful for efficient production of those secondary metabolites in cultivation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1666
New Biotechnology · Volume 31S · July 2014
so as to improve the engineering efficiency, we devised two novel methods termed as “Genome Replication Engineering Assisted Continuous Evolution (GREACE)” and “Stress-InducedMutagenesis Based Adaptive Evolution (SIMBAE)”. Both methods implant controllable in vivo mutagenesis machineries into the cell. Starting the mutagenesis machineries under stressful conditions will trigger continuous mutation generation andsynchronous selection process, and then result a continuous and efficient evolution process which enabled “Mutagenesis coupled-with Selection”. Specifically, GREACE uses a library of activity-compromised proofreading elements of the main DNA polymerase during genome replication as the mutagenesis machinery. In SIMBAE, the mutagenic state is generated by constructing a SIM module which re-produces complex cellular stress-responses implemented by upregulation and down-regulation of various genes. Either method is capable of increasing genomic mutation rate up to 3000-fold. Significantly improved chemical tolerance (n-butanol, acetate, kanamycin), themotolerance, and osmotic tolerance of E. coli were achieved within 9–90 days using the two methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1667
O5-7 Development of two continuous genome engineering strategies for efficient microbial evolution Zhen Cai ∗ , Guodong Luan, Linjiang Zhu, Yin Li Institute of Microbiology, Chinese Academy of Sciences, China
Engineering complex phenotypes of microbes, for instance stress tolerance, remains to be a big challenge in this field. Current evolutionary engineering methods including physical and chemical mutagenesis, global transcription machinery engineering, and artificial transcription factors engineering, use “Mutagenesis followed-by Selection” as the core principle. That is, mutations or genetic perturbations are firstly introduced by exogenous mutagens or genetic manipulations, followed by selection of desired phenotypes. Thus iterative rounds of mutagenesis-selection and frequent manual interventions are often required, resulting in discontinuous and inefficient strain improvements. To address the discontinuity of the existing evolutionary engineering approaches
S24
www.elsevier.com/locate/nbt
with fluorescently labelled B12 . The uptake mechanism remains unclear and is part of our current research. An understanding of transport along the protein shell and its selectivity is an important landmark in our efforts to generate engineered compartments capable of sequestering complex synthetic metabolic pathways.
SYMPOSIUM 5: SYNTHETIC BIOLOGY
O5-5 Novel tuneable gene expression systems based on orthogonal riboswitches Neil Dixon MIB – Manchester, United Kingdom
http://dx.doi.org/10.1016/j.nbt.2014.05.1663
O5-4 Use of transporter plug-ins in building effective microbial cell factories for chemical and fuel production Christopher Grant ∗ , Phattaraporn Morris, Frank Baganz University College London, United Kingdom
Synthetic biology hopes to predictably build cellular factories for the production of chemicals, pharmaceuticals and fuels from cheap renewable feedstocks. A critical, yet understudied, element of this is control of compound transport both into and out of the cell as a means of avoiding issues such as substrate access limitations, substrate and product inhibition and aiding product recovery. We report here a successful strategy for the contextual evaluation of this topic using a library of transport modifying plug-ins combined with multifactorial characterisation. Auxiliary plasmids (pUMP) were used to express a library of transporters as plug-ins alongside two biosynthesis plasmids pGEC41, which oxidises hydrocarbons into fatty alcohols and pADAR7942 which synthesizes bioalkanes from metabolic fatty acid precursors. We demonstrate here benefits of the transporter plug-in approach for: (i) Facilitated delivery hydrophobic substrates to improve whole-cell biocatalysis rates by up to 70 fold (ii) Industrially relevant product yields of over 40 g/Lorganic phase (8 g/Ltotal ) (iii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols (iv) Improving bioalkane synthesis yields from glycerol by >5 fold (v) Reducing alkanol and aldehyde intermediate formation in biosynthesis of bioalkanes by > 10 fold (vi) The integration with in situ product removal strategies to improve bioalkane yields by 10 fold compared to the starting process. This library and plug-in approach is of broad appeal for biological production of hydrophobic compounds and could be a key enabling technology for biological routes for producing a wider range of hydrophobic compounds such as biofuels, fine and specialty chemicals and pharmaceutical intermediates. http://dx.doi.org/10.1016/j.nbt.2014.05.1664
Strategies that permit precisely controlled, differential, and simultaneous expression of multiple genes would be extremely useful for a broad range of metabolic engineering, protein expression and synthetic biology applications. A new paradigm in genetic regulation emerged with the discovery of novel genetic regulatory elements within the 5 UTR of bacterial mRNA. Upon binding to a specific metabolite, these so-called ‘riboswitches’ change conformation, permitting differential gene regulation to occur. As these switches operate via a small molecule-dependent, protein-free mechanism, they present themselves as attractive targets for use as novel genetic control elements. Previously, we showed that it is possible to re-engineer riboswitches so that they no longer bind to their original cognate ligands, but are instead activated by synthetic ligands, and further that these ‘orthogonal’ riboswitches can be used to control heterologous gene expression in vivo [Dixon et al., PNAS 2010]. We have further developed these into multi-component systems that permit fine-tuning over co-expression output stoichiometry, with wide ranging potential applications in functional and structural analysis [Dixon et al., Angew Chem, 2012]. Finally, I will discuss the development of these cellular systems and molecular devices from simply proof-of-principle studies, into the tuneable modular expression system RiboTite, and demonstrate the application of this expression technology for the production of proteins of biotechnological interest. http://dx.doi.org/10.1016/j.nbt.2014.05.1665
O5-6 Signal transduction engineering: a powerful platform technology for enhancing secondary metabolite production Jian-Jiang Zhong ∗ , Yi-Ning Xu, Gao-Yi Tan, Linquan Bai Shanghai Jiao Tong University, China
Streptomycetes and higher fungi produce many bioactive secondary metabolites. Ganoderic acids (GAs) produced by Ganoderma lucidum, a higher fungus, have significant anti-tumor and anti-metastasis activities. Validamycin, an anti-fungal antibiotic produced by Streptomyces hygroscopicus 5008, is an efficient rice sheath blight controller, and can be used as the source for chemical synthesis of voglibos, an antidiabetic drug. Engineering of regulatory mechanism was done to enhance the production of target secondary metabolites. The effects of metal ions on the GAs biosynthesis in liquid cultures of G. lucidum were investigated, and the increased enzyme activities and up-regulation of transcriptional levels of genes in the triterpene biosynthesis were www.elsevier.com/locate/nbt S23
SYMPOSIUM 5: SYNTHETIC BIOLOGY
observed. The regulation mechanism of Mn2+ on the GA biosynthesis was found to be via calcineurin signaling transduction. For the validamycin biosynthesis, our previous study indicated the involvement of A-factor-like cascade. A recent genome-wide analysis reveals three pairs of afsA-arpA in S. hygroscopicus 5008. This work aims to decipher the regulatory role of the multiple afsA-arpA homologs in the A-factor-like cascade, and then to improve the validamycin production by engineering the regulatory cascade. By double deletion of shbR1/R3, the transcriptions of adpA-H and the validamycin biosynthetic genes were up-regulated, and the validamycin production and productivity were enhanced significantly for both the wide-type and a high-producing industrial strain. The transcriptomic analysis revealed that the engineering of A-factorlike signaling cascade caused a shift from primary to secondary metabolism. The signal transduction engineering proposed here is very useful for efficient production of those secondary metabolites in cultivation processes. http://dx.doi.org/10.1016/j.nbt.2014.05.1666
New Biotechnology · Volume 31S · July 2014
so as to improve the engineering efficiency, we devised two novel methods termed as “Genome Replication Engineering Assisted Continuous Evolution (GREACE)” and “Stress-InducedMutagenesis Based Adaptive Evolution (SIMBAE)”. Both methods implant controllable in vivo mutagenesis machineries into the cell. Starting the mutagenesis machineries under stressful conditions will trigger continuous mutation generation andsynchronous selection process, and then result a continuous and efficient evolution process which enabled “Mutagenesis coupled-with Selection”. Specifically, GREACE uses a library of activity-compromised proofreading elements of the main DNA polymerase during genome replication as the mutagenesis machinery. In SIMBAE, the mutagenic state is generated by constructing a SIM module which re-produces complex cellular stress-responses implemented by upregulation and down-regulation of various genes. Either method is capable of increasing genomic mutation rate up to 3000-fold. Significantly improved chemical tolerance (n-butanol, acetate, kanamycin), themotolerance, and osmotic tolerance of E. coli were achieved within 9–90 days using the two methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1667
O5-7 Development of two continuous genome engineering strategies for efficient microbial evolution Zhen Cai ∗ , Guodong Luan, Linjiang Zhu, Yin Li Institute of Microbiology, Chinese Academy of Sciences, China
Engineering complex phenotypes of microbes, for instance stress tolerance, remains to be a big challenge in this field. Current evolutionary engineering methods including physical and chemical mutagenesis, global transcription machinery engineering, and artificial transcription factors engineering, use “Mutagenesis followed-by Selection” as the core principle. That is, mutations or genetic perturbations are firstly introduced by exogenous mutagens or genetic manipulations, followed by selection of desired phenotypes. Thus iterative rounds of mutagenesis-selection and frequent manual interventions are often required, resulting in discontinuous and inefficient strain improvements. To address the discontinuity of the existing evolutionary engineering approaches
S24
www.elsevier.com/locate/nbt