Synthetic cooperation in engineered yeast populations

Abstract

Cooperative interactions are key to diverse biological phenomena ranging from multicellularity to mutualism. Such diversity makes the ability to create and control cooperation desirable for potential applications in areas as varied as agriculture, pollutant treatment, and medicine. Here we show that persistent cooperation can be engineered by introducing a small set of genetic modifications into previously noninteracting cell populations. Specifically, we report the construction of a synthetic obligatory cooperative system, termed CoSMO (cooperation that is synthetic and mutually obligatory), which consists of a pair of nonmating yeast strains, each supplying an essential metabolite to the other strain. The behavior of the two strains in isolation, however, revealed unintended constraints that restrict cooperation, such as asymmetry in starvation tolerance and delays in nutrient release until near cell death. However, the joint system is shown mathematically and experimentally to be viable over a wide range of initial conditions, with oscillating population ratio settling to a value predicted by nutrient supply and consumption. Unexpectedly, even in the absence of explicitly engineered mechanisms to stabilize cooperation, the cooperative system can consistently develop increased ability to survive reductions in population density. Extending synthetic biology from the design of genetic circuits to the engineering of ecological interactions, CoSMO provides a quantitative system for linking processes at the cellular level to the collective behavior at the system level, as well as a genetically tractable system for studying the evolution of cooperation.

Fig. 1.

Rational design of CoSMO. (A) Overproduction of metabolites is required for viable cooperation. At time 0, monocultures of indicated strains grown in synthetic dextrose medium (SD) with the required adenine or lysine supplement (37) were washed free of supplements and mixed. Plots show population dynamics of fluorescent live R (red), fluorescent live Y (green), nonfluorescent dead (gray), and total (black) cells of the coculture as measured by flow cytometry (Methods). (II) Data from three replicate cultures are superimposed. (B) The “wiring” diagram of CoSMO. CoSMO consists of two yeast strains: R_→L^←A, which lacks Ade8 enzyme and harbors Lys21^op enzyme, and Y_→A^←L, which lacks Lys2 enzyme and harbors Ade4^op enzyme. Cells lacking Ade8 (Lys2) cannot synthesize adenine (lysine) and therefore require intake (^←) of the corresponding metabolite. Ade4^op and Lys21^op are no longer sensitive to end-product feed-back inhibition and consequently overproduce (^op) the corresponding metabolite that is eventually released (_→) into the medium (25, 26). Crosses represent genetic inactivation; yellow bars and arrows represent losses and gains in metabolite synthesis, respectively.

Fig. 2.

Characterization of individual strains in monocultures and deduction of CoSMO growth pattern. (A and B) Asymmetry in starvation tolerance between two strains and delayed metabolite release. At time 0, monocultures of the two strains grown in the presence of the required supplement were washed free of the supplement. (A) Live population density over time for an initial population density of ≈3 × 10⁵ cells per ml. (B) Dead population density (Upper) and the concentration of lysine or adenine released into the medium over time (Lower) as measured by a bioassay (Methods) for an initial population density of ≈6 × 10⁶ cells per ml. The left and right scales are for experiments on Y_→A^←L (squares) and R_→L^←A (circles), respectively. Gray vertical lines mark the time T_I when residual growth ends and the time T_R when R_→L^←A enters death phase and releases lysine. (C) A schematic diagram of the initial stage of CoSMO growth deduced from A and B. R and Y denote live population densities of R_→L^←A and Y_→A^←L, respectively. Their initial values R₀ and Y₀ increase I_R- and I_Y-fold, respectively, during residual growth until time T_I. After T_I, adenine released from dying Y_→A^←L enables growth of R_→L^←A. By time ≈T_R, most of the Y_→A^←L population has died and R is at a local maximum R_max. Lysine is subsequently released from dying R_→L^←A, and at some time τ after T_R, results in an increase in Y under conditions that permit CoSMO viability. The death rate for R_→L^←A after T_R is D_R, and for Y_→A^←L is D_Y from T_I to T_R and D_YLate from T_R onward. The total cell density, which is the sum of R, Y, and dead populations, consequently takes on a pattern of “rise-plateau-rise,” with each rise resulting from net growth of at least one partner.

Fig. 3.

Phase diagram for CoSMO viability. The domain of viability for CoSMO at volume V of 2.6 ml is bounded by a black vertical line (single arrowhead, Appendix, inequality 6) and gray curves (Appendix, Inequality 8, with I_Y set to different values in the experimentally observed range from 2 to 4). The shoulder (⌉) represents the viability threshold imposed by the density requirement alone (Appendix, Eqs. 4 and 5) and is therefore not affected by the culture volume. Different volumes affect only the black vertical line (single arrowhead) and the horizontal asymptote (double arrowhead), which shift along the R₀ and Y₀ axis according to the initial-number requirements expressed in Appendix inequalities 6 and 8a, respectively. Circles indicate values of (R₀, Y₀) corresponding to different experiments (orange for Fig. 1AII; purple from top left to bottom right for Fig. 4A, from I to VI; and brown for Fig. 4B). In experiment marked with cyan, one of five replicate cultures was viable (time series not shown); in experiments marked with black, zero of four or five replicate cultures was viable (time series not shown). Overall, the viability–inviability outcome of replicate CoSMO cultures close to the calculated boundary is highly variable (open circles), whereas cultures significantly above and below the boundary show 100% viability (filled circles) and 0% viability (broken circles), respectively.

Fig. 4.

Viability of CoSMO. Monocultures of the two strains were washed free of adenine and lysine, and mixed at time 0. (A) CoSMO is viable under a wide range of initial partner ratios. The two strains were mixed at the indicated R_→L^←A:Y_→A^←L ratios (R₀:Y₀) and at the same total initial cell density and culture volume (≈5 × 10⁵ cells per ml × 2.6 ml = 1.3 × 10⁶ cells per culture, four replicate cultures per condition). Plots show culture turbidity in OD₆₀₀ (optical density at 600 nm) over time. OD₆₀₀ of 1 corresponds to a population density from 1 × 10⁷ to 5 × 10⁷ cells per ml depending on the cell size. (B) Stochastic CoSMO behavior close to the initial-density requirement. Three replicate cultures (≥20 ml) were set up at 1.1 × 10⁴ cells per ml per strain. Plots show the dynamics of live R_→L^←A (red), live Y_→A^←L (green), dead (gray), and total (black) cell densities. One of the cultures was inviable (I), whereas the other two were viable (II and III).

Fig. 5.

Long-term changes in CoSMO. (A) Stabilization of partner ratios. At time 0, duplicate CoSMO cultures (brown and blue) were initiated at OD₆₀₀ of 0.01 (4.7 × 10⁵ total cells per ml) and R_→L^←A:Y_→A^←L ratios of 10³ (I), 1 (II), and 10⁻³ (III). When OD₆₀₀ exceeded the set point of 0.06 for the first time, two 3-ml samples were taken from each culture (brown and magenta from the brown; blue and cyan from the blue), and thereafter diluted once per day (magenta and cyan) or twice per day (brown and blue) to the set point. A low set point was chosen so that nutrients other than adenine and lysine were not limiting. Plots show R_→L^←A:Y_→A^←L ratios over time, with triangles marking points of dilution. (B) Increased ability to survive reductions in population density. Five 2.6-ml CoSMO cultures, initiated at different partner ratios, were grown to near-saturation and used as Round-0 cultures for five independent series. After 10 rounds of dilution and regrowth in 2.6 ml, a near-saturation Round-10 culture was obtained for each series. Each row corresponds to a particular series and depicts the number of tubes (of three) that were viable at indicated dilutions for Round-0 (Left) and Round-10 (Right) cultures. Population densities of R_→L^←A (red) and Y_→A^←L (green) in million cells per ml for Round-0 and Round-10 cultures are shown in the Inset.