Synthetic microbial consortia that can mimic natural systems have the potential to become a powerful biotechnology for various applications. One highly desirable feature of these consortia is that they can be precisely regulated. In this work we designed a programmable, symbiotic circuit that enables continuous tuning of the growth rate and composition of a synthetic consortium. We implemented our general design through the cross-feeding of tryptophan and tyrosine by two E. coli auxotrophs. By regulating the expression of genes related to the export or production of these amino acids, we were able to tune the metabolite exchanges and achieve a wide range of growth rates and strain ratios. In addition, by inverting the relationship of growth/ratio vs. inducer concentrations, we were able to “program” the co-culture for pre-specified attributes with the proper addition of inducing chemicals. This programmable proof-of-concept circuit or its variants can be applied to more complex systems where precise tuning of the consortium would facilitate the optimization of specific objectives, such as increasing the overall efficiency of microbial production of biofuels or pharmaceuticals.
References
[1]
Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, et al. (2007) The Human Microbiome Project. Nature 449: 804–810.
[2]
Daims H, Taylor MW, Wagner M (2006) Wastewater treatment: a model system for microbial ecology. Trends in Biotechnology 24: 483–489.
[3]
Eiteman MA, Lee SA, Altman E (2008) A co-fermentation strategy to consume sugar mixtures effectively. J Biol Eng 2: 3.
[4]
Brenner K, You L, Arnold FH (2008) Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol 26: 483–489.
[5]
Basu S, Gerchman Y, Collins CH, Arnold FH, Weiss R (2005) A synthetic multicellular system for programmed pattern formation. Nature 434: 1130–1134.
[6]
Shou WY, Ram S, Vilar JMG (2007) Synthetic cooperation in engineered yeast populations. Proceedings of the National Academy of Sciences of the United States of America 104: 1877–1882.
[7]
Weber W, Daoud-El Baba M, Fussenegger M (2007) Synthetic ecosystems based on airborne inter- and intrakingdom communication. Proceedings of the National Academy of Sciences of the United States of America 104: 10435–10440.
[8]
Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4: 187.
[9]
Brenner K, Karig DK, Weiss R, Arnold FH (2007) Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium. Proceedings of the National Academy of Sciences of the United States of America 104: 17300–17304.
[10]
Brenner K, Arnold FH (2011) Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium. PLoS One 6:
[11]
Doroshenko V, Airich L, Vitushkina M, Kolokolova A, Livshits V, et al. (2007) YddG from Escherichia coli promotes export of aromatic amino acids. FEMS Microbiol Lett 275: 312–318.
[12]
Pittard J, Camakaris H, Yang J (2005) The TyrR regulon. Molecular Microbiology 55: 16–26.
[13]
Aiba S, Tsunekawa H, Imanaka T (1982) New approach to tryptophan production by Escherichia coli - genetic manipulation of composite plasmids in vitro. Applied and Environmental Microbiology 43: 289–297.
[14]
Azuma S, Tsunekawa H, Okabe M, Okamoto R, Aiba S (1993) Hyper-production of L-tryptophan via fermentation with crystallization. Applied Microbiology and Biotechnology 39: 471–476.
[15]
Bongaerts J, Kramer M, Muller U, Raeven L, Wubbolts M (2001) Metabolic engineering for microbial production of aromatic amino acids and derived compounds. Metabolic Engineering 3: 289–300.
[16]
Caligiuri MG, Bauerle R (1991) Identification of amino acid residues involved in feedback-regulation of the Anthranilate synthase complex from Salmonella typhimurium - evidence for an amino-terminal regulatory site. Journal of Biological Chemistry 266: 8328–8335.
[17]
Spraggon G, Kim C, Nguyen-Huu X, Yee MC, Yanofsky C, et al. (2001) The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan. Proc Natl Acad Sci U S A 98: 6021–6026.
[18]
Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72: 211–222.
[19]
Lee SK, Chou HH, Pfleger BF, Newman JD, Yoshikuni Y, et al. (2007) Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters. Applied and Environmental Microbiology 73: 5711–5715.
[20]
Lee SK, Keasling JD (2005) A propionate-inducible expression system for enteric bacteria. Appl Environ Microbiol 71: 6856–6862.
[21]
Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, et al. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotech 20: 87–90.
[22]
Harcombe W (2010) Novel cooperation experimentally evolved between species. Evolution; international journal of organic evolution 64: 2166–2172.
[23]
Hillesland KL, Stahl DA (2010) Rapid evolution of stability and productivity at the origin of a microbial mutualism. Proc Natl Acad Sci U S A 107: 2124–2129.
[24]
Fu N, Peiris P, Markham J, Bavor J (2009) A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures. Enzyme and Microbial Technology 45: 210–217.
[25]
Eiteman MA, Lee SA, Altman R, Altman E (2009) A Substrate-Selective Co-Fermentation Strategy With Escherichia coli Produces Lactate by Simultaneously Consuming Xylose and Glucose. Biotechnology and Bioengineering 102: 822–827.
[26]
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.
[27]
Elowitz MB, Levine AJ, Siggia ED, Swain PS (2002) Stochastic gene expression in a single cell. Science 297: 1183–1186.