Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production
Introduction
Developments in the construction of biosynthetic pathways in microorganisms have allowed fermentation technology to be considered as an increasingly viable means of chemical production (Atsumi et al., 2008, Dellomonaco et al., 2011, Martin et al., 2003, Qian et al., 2009, Steen et al., 2010, Yim et al., 2011). However, several synthetic metabolic pathways are directly competing for usage of endogenous metabolites with microbial cell growth (Atsumi et al., 2008, Hanai et al., 2007, Jun Choi et al., 2012, Rodriguez et al., 2014). Additionally, overexpression of the enzymes in these pathways and/or overproduction of toxic intermediates occasionally causes growth retardation of the host microorganisms (Ajikumar et al., 2010, Nakamura and Whited, 2003, Rathnasingh et al., 2012). In short, the optimal metabolic state for target chemical production often directly conflicts with bacterial cell growth (Gadkar et al., 2005). For an economically viable fermentation and further improvement to chemical production, novel engineering strategies to manage this tradeoff relationship must be utilized.
One of the major goals in synthetic biology is to design synthetic gene networks to reprogram cell behavior as desired for practical applications such as microbial fermentation (Khalil and Collins, 2010). Several synthetic genetic circuits have been designed and constructed in pathway engineering for improved productivity and yield by controlling metabolic flux of critical metabolic intermediates (e.g., lycopene production using an acetyl-phosphate sensor (Farmer and Liao, 2000), fatty acyl acid ester production based on a dynamic sensor-regulator of fatty acyl-CoA (Zhang et al., 2012), gluconate production using a dynamic metabolic valve (Solomon et al., 2012)). Furthermore, some genetic circuits have been designed for conditional switching of target gene expression by addition of exogenous chemical inducers, such as the fatty acid synthesis via dynamic control of fatty acid elongation (Torella et al., 2013). We also have presented a synthetic genetic circuit, a metabolic toggle switch (MTS) controlling conditional redirection of metabolic flux from endogenous pathways toward targeted synthetic metabolic pathway (Soma et al., 2014). This flux regulation system significantly improved the isopropanol production of an engineered Escherichia coli strain by drastically switching the metabolic flux from cell mass development to production of the target compound. It required strict timing of the addition of exogenous chemical inducer in order to achieve its desired function of ensuring adequate cell mass and improvement in productivity of the target compound. To decrease such difficulty, ideally, the engineered microbes would sense their population, and in the response to reaching appropriate cell density, self-trigger the expression of the appropriate pathway genes for efficient target compound production.
Quorum sensing (QS) systems have been found in several microbial species as a sensor-regulator system that detects local cell population density and triggers expression of particular genes for coordinated collective cell behavior in response to a cell density threshold (Miller and Bassler, 2001). One of the most studied QS systems is the lux system in Vibrio fischeri which harbors the luminescence (lux) operon (Egland and Greenberg, 1999, Fuqua and Greenberg, 2002). The lux system has been reconstituted in heterogeneous hosts, such as E. coli, to investigate its dynamic behavior (Haseltine and Arnold, 2008) and to design synthetic cell–cell communication systems (Balagaddé et al., 2008, Danino et al., 2010). Furthermore, the lux system, as well as other QS systems, has been applied to synthetic genetic circuits facilitating protein production (Kobayashi et al., 2004, Pai et al., 2012, Shong and Collins, 2014, Shong et al., 2013, Tsao et al., 2010). However, no examples of QS applied to microbial chemical production have hitherto been published. This situation may be because the threshold cell density (cell density for QS dependent induction of gene expression) of the almost all synthetic or native QS systems is fixed at a quite low value (Haseltine and Arnold, 2008). Microbial fermentation requires optimal induction of target metabolic pathways at appropriate cell densities, which may differ among the diverse target compounds.
In this study, for the multi-purpose application of QS as a sensor-regulator system in metabolic engineering, we developed a synthetic lux system by combination of a synthetic lux promoter and a positive feedback loop. This QS system׳s threshold cell density can be tuned depending on the concentration of exogenous inducer, isopropyl β-d-1-thiogalactopyranoside (IPTG) added at the beginning of the fermentation. This enables self-induction of target gene expression at a desired cell density. Additionally, we applied this system to trigger a metabolic toggle switch (MTS) in order to demonstrate the self-induced redirection of metabolic flux from the TCA cycle toward isopropanol production at a desired E. coli density. Isopropanol is one of the simplest secondary alcohols that can be dehydrated to yield propylene, a monomer for making polypropylene currently derived from petroleum. As polypropylene is currently used as a material for many industrial products, it is expected that world demand for propylene will continue to grow in the future. We have previously engineered a synthetic metabolic pathway and E. coli strains for microbial isopropanol production from inexpensive and renewable feedstock, such as biomass-derived saccharides (Hanai et al., 2007, Inokuma et al., 2010, Soma et al., 2012).
Section snippets
Chemicals and reagents
All chemicals were purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan) unless otherwise specified. Restriction enzymes and phosphatase were from New England Biolabs (Ipswich, MA, USA), ligase (rapid DNA ligation kit) was from Roche (Manheim, Germany), and KOD Plus Neo DNA polymerase was from TOYOBO Co., Ltd. (Osaka, Japan). Oligonucleotides were synthesized by Life Technologies Japan Ltd. (Tokyo, Japan). N-(β-ketocaproyl)-L-Homoserine lactone (AHL) was purchased from Sigma-Aldrich
Characterization of a synthetic lux promoter and a reconstituted lux regulon
The lux system-mediated detection of cell density requires the interaction of a diffusible auto-inducer acylhomoserine lactone (AHL) and AHL-dependent transcription activator LuxR (Fuqua and Greenberg, 2002). AHL is produced by LuxI under the control of the lux promoter (Egland and Greenberg, 1999) activated by the AHL–LuxR complex. All cells in the population produce AHL at a low basal level that can freely diffuse from the cell. When extracellular AHL reaches the threshold concentration; it
Conclusion
We present a tunable cell density sensor-regulator by reconstituting the lux system. This system enables a predetermined threshold cell density to trigger expression of the target genes. As the result, this QS system can be used in many types of fermentations to induce chemical production pathways and other targeted genes expression. Accordingly, we show that QS-signal transduction can be adjusted by cooperation of LacI repressor and synthetic lux promoter through design of synthesized operator
Acknowledgments
We thank J. Hasty (University of California, San Diego) for providing the gene clock plasmid pTD103luxI/GFP, F. Matsuda (Osaka University) for measuring intracellular metabolites using LC-QqQ-MS and GC-Q-MS analysis, J. T. McEwen (University of California, Davis) for language help, and S. Atsumi (University of California, Davis) for helpful advice. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (23119002) from the Ministry of Education, Culture, Sports,
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