Synthetic control of a fitness tradeoff in yeast nitrogen metabolism

Abstract

Background: Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology.

Results: Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments.

Conclusion: The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities.

Figure 1

Schematic of ammonia assimilation in S. cerevisiae. Ammonia is transported into the cell by three transporters, Mep1p, Mep2p, and Mep3p. Three enzymes, Gdh1p, Glt1p, and Gdh3p, convert ammonia to glutamate and glutamine. Gdh1p is responsible for the majority of ammonia assimilation. Gdh1p is regulated by four transcription factors, Gat1p, Gln3p, Dal80p, and Gzf3p.

Figure 2

Characterization of mutant strains with different Gdh1p noise and abundance profiles indicates that fitness in different ammonia conditions correlates with noise in Gdh1p expression. (A) Schematic of the genetic construct for building the GDH1 promoter mutant library. A region 500 nt upstream of the GDH1 coding sequence was amplified by mutagenic PCR, assembled with a selectable LEU2 marker, and reintegrated to construct a mutant library. (B) A random set of clones from the GDH1 promoter mutant library exhibit different values for Gdh1p mean abundance and noise. Noise is calculated as the square of the coefficient of variation (CV²). Sets of mutants with low, wildtype, and high mean abundances are indicated as blue, red, and green, respectively. Wildtype is indicated in yellow. All errors are within 5% of the reported values. (C) Relative fitness in high and low ammonia concentrations correlates with noise in Gdh1p expression (correlation coefficient = 0.83, R² = 0.69). Relative fitness for select constant abundance-different noise mutant sets is reported as W_env, or the ratio of fitness in 556 mM ammonia to fitness in 17 mM ammonia using the competition fitness assays. The wildtype relative fitness is equal to 1. Low Gdh1p noise is correlated with high fitness in ammonia-poor conditions and low fitness in ammonia-rich conditions (W_env < 1). High Gdh1p noise is correlated with low fitness in ammonia-poor conditions and high fitness in ammonia-rich conditions (W_env > 1). (D) Relative fitness in high and low ammonia concentrations does not correlate with Gdh1p abundance (correlation coefficient = 0.079, R² = 0.0062). Relative fitness (W_env) for a set of GDH1 promoter mutants with similar Gdh1p noise values (approximate CV² = 0.65) and different abundance values is reported.

Figure 3

Variation in Gdh1p expression provides different growth trends in high and low ammonia environments. (A) Flow cytometry histograms of two GDH1 promoter mutants exhibiting different noise and similar abundance profiles in Gdh1p as the wildtype strain. The 'high noise' mutant has a square of the coefficient of variation (σ²/p²) of 0.74 (20% higher than wildtype) and the 'low noise' mutant has a σ²/p² of 0.56 (10% lower than wildtype). (B) The high noise mutant exhibits greater resistance to ammonia stress under high ammonia concentrations. The fold change in CFUs is reported at different time points following exposure to high ammonia (600 mM) media using the plate-based fitness assays. Dashed line, wildtype; gray line, high noise strain; black line, low noise strain. (C) The high noise mutant exhibits greater delayed toxicity to ammonia stress under high ammonia and low potassium concentrations. The fold change in CFUs is reported at different time points following exposure to high ammonia (600 mM) and low potassium (17 mM) media. Dashed line, wildtype; gray line, high noise strain; black line, low noise strain. (D) The low noise mutant exhibits greater fitness in low ammonia environments. Fitness for the high and low noise strains were measured across a range of low ammonia concentrations using the competition fitness assays. Fitness is reported as the natural log of the change in frequency over the growth period relative to the wildtype strain. Black circles, low noise strain; gray circles, high noise strain.

Figure 4

Synthetic control of Dal80p levels tunes noise in Gdh1p expression. (A) Schematic of the genetic construct for building the galactose-tunable Dal80p system. A region 500 nt upstream of the DAL80 coding region was replaced through homologous recombination with a construct encoding the GAL1-10 promoter and a selectable LEU2 marker. (B) DAL80 transcript levels vary linearly with exogenous galactose in the engineered strain. Relative DAL80 transcript levels were measured by qRT-PCR and are shown relative to wildtype DAL80 transcript levels. (C) Gdh1p abundance does not change as Dal80p levels change. The percent galactose added to the culture is shown in parentheses. (D) Noise in Gdh1p expression changes as Dal80p levels change. Populations of the engineered strain show higher noise at low Dal80p levels (low galactose concentrations) and lower noise with increasing Dal80p (increasing galactose concentrations). Percent galactose added to the culture is shown in parentheses.

Figure 5

Synthetic control of Dal80p allows for tuning of environment-dependent fitness. (A) The fitness of the engineered strain displays varying trends with ammonia concentrations at different Dal80p expression levels. Dal80p expression levels were varied by altering the concentration of galactose in the media. Fitness was measured at the indicated ammonia concentrations using the competition fitness assays and is reported as in Figure 3d. (B) The fitness of the engineered strain does not change with varying concentrations of other nitrogen sources at different Dal80p expression levels. Dal80p expression levels were varied by altering the concentration of galactose in the media. Fitness was measured at the indicated ammonia concentrations using the competition fitness assays and is reported as in Figure 3d. (C) Relative fitness in high and low ammonia concentrations is tuned through the exogenous addition of galactose to the engineered strain. Relative fitness (W_env) is reported as the ratio of fitness in 556 mM ammonia to fitness in 8.7 mM ammonia using the competition fitness assays. Low galactose concentrations tune the strain to exhibit greater fitness in ammonia-rich conditions than ammonia-poor conditions (W_env > 1), whereas high galactose concentrations tune the strain to exhibit greater fitness in ammonia-poor conditions that ammonia-rich conditions (W_env < 1).