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A programmable Escherichia coli consortium via tunable symbiosis.

Kerner A, Park J, Williams A, Lin XN - PLoS ONE (2012)

Bottom Line: We implemented our general design through the cross-feeding of tryptophan and tyrosine by two E. coli auxotrophs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
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.

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Basic schematic of the tunable cross-feeding circuit.(A) In this general design, inducer 1 and inducer 2 control the export of metabolites 1 and 2, respectively. The two auxotrophs must cross-feed in order to survive in the minimal medium. (B) In our specific implementation, two E. coli auxotrophic strains exchange tryptophan (Trp) and tyrosine (Tyr). The forced symbiosis is controlled by plasmids pAK1 (in the Trp auxotroph, W3) and pAK5 (in the Tyr auxotroph, Y3). Plasmid pAK1 contains gene yddG behind the tunable promoter PBAD, and pAK5 contains trpEDfbr behind PprpB (Methods). Strain Y3 is tagged with yellow fluorescent protein (YFP).
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pone-0034032-g001: Basic schematic of the tunable cross-feeding circuit.(A) In this general design, inducer 1 and inducer 2 control the export of metabolites 1 and 2, respectively. The two auxotrophs must cross-feed in order to survive in the minimal medium. (B) In our specific implementation, two E. coli auxotrophic strains exchange tryptophan (Trp) and tyrosine (Tyr). The forced symbiosis is controlled by plasmids pAK1 (in the Trp auxotroph, W3) and pAK5 (in the Tyr auxotroph, Y3). Plasmid pAK1 contains gene yddG behind the tunable promoter PBAD, and pAK5 contains trpEDfbr behind PprpB (Methods). Strain Y3 is tagged with yellow fluorescent protein (YFP).

Mentions: We have designed a genetic circuit, based on metabolic cross feeding, to enable continuous tuning of the growth rate and composition of a synthetic two-member microbial consortium. A schematic of our tunable circuit is shown in Figure 1A, wherein two auxotrophs are forced to depend upon each other for growth. In a minimal medium lacking key metabolites, these two microbes do not grow unless they exchange the required nutrients in an efficient manner. Previous work suggested that such a pair of inter-dependent microbes, when grown together in a batch co-culture and given enough time, reaches a pseudo steady state characterized by unchanging concentrations of cross-fed metabolites and consortium composition (Reppas, Lin, et al., manuscript in preparation). Serial passaging experiments with auxotroph pairs indicated that this was indeed the case (data not shown). At this pseudo steady state the two consortium members have the same growth rate. In addition, this growth rate of the co-culture and the ratio of the two microbes are determined solely by each auxotroph's export rate of its partner's required metabolite and the auxotroph's growth requirement for the metabolite it demands, as illustrated mathematically below (see Methods for details):(1)(2)where μ represents the co-culture growth rate, r is the ratio of the cell density of Auxotroph 2 (n2) versus that of Auxotroph 1 (n1). α1 denotes Auxotroph 1's export rate of Metabolite 2 required by Auxotroph 2 (e.g. with a unit of mmol/gDM*hr) and β1 is Auxotroph 1's cellular requirement for Metabolite 1 (e.g. with a unit of mmol/gDM). Similarly, α2 and β2 describe Auxotroph 2's corresponding properties. Based on this theoretical prediction, it should therefore be possible to control the co-culture growth rate and the ratio of the two microbes by manipulating either the auxotrophs' export of the two cross-fed metabolites (i.e. α1 and α2) or their cellular requirement for the metabolites (i.e. β1 and β2). The former strategy appeared more straightforward and we further decided to explore the usage of chemical inducers to regulate the synthesis and transport pathways related to the export of the two cross-fed metabolites (Figure 1A).


A programmable Escherichia coli consortium via tunable symbiosis.

Kerner A, Park J, Williams A, Lin XN - PLoS ONE (2012)

Basic schematic of the tunable cross-feeding circuit.(A) In this general design, inducer 1 and inducer 2 control the export of metabolites 1 and 2, respectively. The two auxotrophs must cross-feed in order to survive in the minimal medium. (B) In our specific implementation, two E. coli auxotrophic strains exchange tryptophan (Trp) and tyrosine (Tyr). The forced symbiosis is controlled by plasmids pAK1 (in the Trp auxotroph, W3) and pAK5 (in the Tyr auxotroph, Y3). Plasmid pAK1 contains gene yddG behind the tunable promoter PBAD, and pAK5 contains trpEDfbr behind PprpB (Methods). Strain Y3 is tagged with yellow fluorescent protein (YFP).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316586&req=5

pone-0034032-g001: Basic schematic of the tunable cross-feeding circuit.(A) In this general design, inducer 1 and inducer 2 control the export of metabolites 1 and 2, respectively. The two auxotrophs must cross-feed in order to survive in the minimal medium. (B) In our specific implementation, two E. coli auxotrophic strains exchange tryptophan (Trp) and tyrosine (Tyr). The forced symbiosis is controlled by plasmids pAK1 (in the Trp auxotroph, W3) and pAK5 (in the Tyr auxotroph, Y3). Plasmid pAK1 contains gene yddG behind the tunable promoter PBAD, and pAK5 contains trpEDfbr behind PprpB (Methods). Strain Y3 is tagged with yellow fluorescent protein (YFP).
Mentions: We have designed a genetic circuit, based on metabolic cross feeding, to enable continuous tuning of the growth rate and composition of a synthetic two-member microbial consortium. A schematic of our tunable circuit is shown in Figure 1A, wherein two auxotrophs are forced to depend upon each other for growth. In a minimal medium lacking key metabolites, these two microbes do not grow unless they exchange the required nutrients in an efficient manner. Previous work suggested that such a pair of inter-dependent microbes, when grown together in a batch co-culture and given enough time, reaches a pseudo steady state characterized by unchanging concentrations of cross-fed metabolites and consortium composition (Reppas, Lin, et al., manuscript in preparation). Serial passaging experiments with auxotroph pairs indicated that this was indeed the case (data not shown). At this pseudo steady state the two consortium members have the same growth rate. In addition, this growth rate of the co-culture and the ratio of the two microbes are determined solely by each auxotroph's export rate of its partner's required metabolite and the auxotroph's growth requirement for the metabolite it demands, as illustrated mathematically below (see Methods for details):(1)(2)where μ represents the co-culture growth rate, r is the ratio of the cell density of Auxotroph 2 (n2) versus that of Auxotroph 1 (n1). α1 denotes Auxotroph 1's export rate of Metabolite 2 required by Auxotroph 2 (e.g. with a unit of mmol/gDM*hr) and β1 is Auxotroph 1's cellular requirement for Metabolite 1 (e.g. with a unit of mmol/gDM). Similarly, α2 and β2 describe Auxotroph 2's corresponding properties. Based on this theoretical prediction, it should therefore be possible to control the co-culture growth rate and the ratio of the two microbes by manipulating either the auxotrophs' export of the two cross-fed metabolites (i.e. α1 and α2) or their cellular requirement for the metabolites (i.e. β1 and β2). The former strategy appeared more straightforward and we further decided to explore the usage of chemical inducers to regulate the synthesis and transport pathways related to the export of the two cross-fed metabolites (Figure 1A).

Bottom Line: We implemented our general design through the cross-feeding of tryptophan and tyrosine by two E. coli auxotrophs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus