Synthetic Escherichia coli consortia engineered for syntrophy demonstrate enhanced biomass productivity

Abstract

Synthetic Escherichia coli consortia engineered for syntrophy demonstrated enhanced biomass productivity relative to monocultures. Binary consortia were designed to mimic a ubiquitous, naturally occurring ecological template of primary productivity supported by secondary consumption. The synthetic consortia replicated this evolution-proven strategy by combining a glucose positive E. coli strain, which served as the system's primary producer, with a glucose negative E. coli strain which consumed metabolic byproducts from the primary producer. The engineered consortia utilized strategic division of labor to simultaneously optimize multiple tasks enhancing overall culture performance. Consortial interactions resulted in the emergent property of enhanced system biomass productivity which was demonstrated with three distinct culturing systems: batch, chemostat and biofilm growth. Glucose-based biomass productivity increased by ∼15, 20 and 50% compared to appropriate monoculture controls for these three culturing systems, respectively. Interestingly, the consortial interactions also produced biofilms with predictable, self-assembling, laminated microstructures. This study establishes a metabolic engineering paradigm which can be easily adapted to existing E. coli based bioprocesses to improve productivity based on a robust ecological theme.

Figure 1

Preferential acetate catabolism by glucose negative strain 403G100 cultured in M9 medium containing glucose and sodium acetate. Strain 403G100 did not grow in M9 medium containing glucose as the sole electron donor. Error bars represent ± 1 standard deviation from two independent shake flask experiments.

Figure 2

Binary consortium and monoculture batch growth. A) Binary consortium (403G100 + WT) and wild-type monoculture batch growth glucose and biomass concentration time profiles. All experiments were performed with M9 medium supplemented with glucose as the sole reduced carbon source. Error bars represent ± 1 standard deviation from three independent batch experiments. B) Strain 403G100 monocultures did not demonstrate significant growth or glucose consumption. Error bars represent ± 1 standard deviation from three technical replicates of measurements.

Figure 3

Binary consortium (403G100 + WT) and monoculture batch growth physiological parameters. A) Biomass yield on glucose (g CDW/g glucose) for the engineered binary consortium and wild-type monoculture control. Yields calculated from exponential growth phase data. Glucose negative strain 403G100 did not produce significant growth on glucose. Error bars represent ± 1 standard deviation from three independent batch experiments. B) Exponential growth phase acetate accumulation plotted as a function of biomass from a representative batch growth experiment. Error bars represent ± 1 standard deviation from three technical replicates of measurements.

Figure 4

Binary consortium (403G100 + WT) and monoculture biomass concentrations during glucose-limited chemostat cultivation. Cultures were grown continuously in glucose-limited chemostats (D= 0.1/hr) under two different aeration regimes (high-aeration = 1 L/min, low-aeration = 0.15 L/min air sparge). Strain 403G100 monocultures had negligible steady-state biomass concentrations (below detection limit of 0.01 g CDW/L) for both aeration regimes. Error bars represent ± 1 standard deviations from three independent chemostat experiments.

Figure 5

Binary consortium (403G100 + WT) and monoculture chemostat specific glucose uptake and acetate secretion rates. Cultures were grown continuously in glucose-limited chemostats (D= 0.1/hr) under two different aeration regimes (high-aeration = 1L/min, low-aeration = 0.3 L/min). High-aeration data error bars represent ± 1 standard deviation from three independent chemostat experiments. Low-aeration data error bars represent ± 1 standard deviation from three technical measurements of a representative data set. The low-aeration cultures were highly sensitive to small differences in the rotameter controlled sparge rate. All three low-aeration chemostat experiments exhibited the presented trends.

Figure 6

Colony forming units per biofilm (CFUs/biofilm) for the binary consortium (403G100 + WT) and monoculture controls. Biofilms were cultured on M9 agar containing 1% (w/v) glucose as the sole reduced carbon source. CFUs were enumerated on non-selective (LB) agar. The binary consortium produced more CFUs/biofilm than the sum of monocultures. Error bars represent ± 1 standard deviation from at least three independently cultured biofilms.

Figure 7

Culturing properties of a dual engineered binary consortium comprised of glucose positive strain 307G100 (ΔaceAΔldhAΔfrdA ) and glucose negative strain 403G100. A) Biomass and glucose concentration time profiles for dual engineered binary consortium and monoculture control during batch shake flask cultivation. Data collected from four independent shake flask experiments with different sampling intervals. B) Colony biofilm cultivation data for the dual engineered binary consortium and appropriate monoculture controls grown on M9 medium with glucose as the sole reduced carbon source. Error bars represent ± 1 standard deviation from three independently cultured biofilms.

Figure 8

Epi-fluorescence micrographs and quantitative image analysis of dual engineered binary consortium (403G100 + 307G100) and strain 307G100 monoculture colony biofilms. A) Dual engineered binary consortium biofilm micrograph with glucose negative strain 403G100 expressing reporter protein td-tomato (red) and glucose positive strain 307G100 expressing reporter protein m-citrine (green). Biofilm image oriented with air-interface on left. Glucose negative strain 403G100 localized primarily at the air interface; red cells at the membrane interface are a result of daily aerobic biofilm plate transfers. Dark regions within biofilm are an artifact of cryosectioning thick biofilms. B) Micrograph of control biofilm comprised of two 307G100 strains each expressing a different fluorescent protein (td-tomato, m-citrine). C) Average fluorescence intensity of red and green reporter proteins as a function of position within biofilm for the dual engineered consortium. Depth is measured from the air interface into the biofilm. D) Average fluorescence intensity as a function of position within the biofilm for strain 307G100 expressing either a red or green fluorescence reporter protein. Fluorescence intensity versus position data is the mean of four line segments chosen at random biofilm positions. Error bars represent ± 1 standard deviation.