Design of stable and self-regulated microbial consortia for chemical synthesis

Abstract

Microbial coculture engineering has emerged as a promising strategy for biomanufacturing. Stability and self-regulation pose a significant challenge for the generation of intrinsically robust cocultures for large-scale applications. Here, we introduce the use of multi-metabolite cross-feeding (MMCF) to establish a close correlation between the strains and the design rules for selecting the appropriate metabolic branches. This leads to an intrinicially stable two-strain coculture where the population composition and the product titer are insensitive to the initial inoculation ratios. With an intermediate-responsive biosensor, the population of the microbial coculture is autonomously balanced to minimize intermediate accumulation. This static-dynamic strategy is extendable to three-strain cocultures, as demonstrated with de novo biosynthesis of silybin/isosilybin. This strategy is generally applicable, paving the way to the industrial application of microbial cocultures.

Fig. 1. Design principles of the stable coculture system.

The two strains are engineered to utilize glycerol and glucose, respectively, and cross-feed each other with amino acids and the TCA cycle intermediates. Blue crosses denote gene knockouts to block glucose/glycerol utilization; red crosses denote gene knockouts to block glutamate biosynthesis and the TCA cycle. MMCF multi-metabolite cross-feeding. Genes: glK encodes glucokinase, ptsG encodes PTS glucose transport protein, manXYZ encodes mannose permease, pykA/F encodes pyruvate kinase, ppc encodes phosphoenolpyruvate carboxylase, glpK encodes glycerol kinase, gdhA encodes glutamate dehydrogenase, gltBD encodes glutamate synthase. Metabolites: PEP phosphoenolpyruvate, PYR pyruvate, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids.

Fig. 2. Curves of cell growth and population composition in the three coculture systems.

a The Neutralistic coculture; b the Commensalistic coculture; c the Mutualistic coculture. IIRs, the initial inoculation ratios. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 3. Efficient salidroside production and scale-up using coculture engineering.

a Schematic of the E. coli-E. coli cocultures to accommodate the salidroside biosynthetic pathway and convert a glycerol and glucose mixture to salidroside (The salidroside biosynthetic pathway is shown in orange. For the detailed pathway, see Supplementary Fig. 5). Curves of cell growth and population change in b the Neutralistic Bgly1-Tyr/Bglc1-Sal coculture, c the Commensalistic Bgly1-Tyr/Bglc2-Sal coculture, and d the Mutualistic Bgly2-Tyr/Bglc2-Sal coculture. e Comparison of salidroside titers in the three coculture systems. f Scale-up production of salidroside using the Bgly2-Tyr/Bglc2-Sal coculture. MMCF multi-metabolite cross-feeding, IIRs the initial inoculation ratios. Metabolites: PEP phosphoenolpyruvate, G6P glucose-6-phosphate, UDPG uridine diphosphate glucose, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 4. Production of coniferol using coculture engineering.

a Schematic of the E. coli-E. coli cocultures to accommodate the coniferol biosynthetic pathway and convert a glycerol and glucose mixture to coniferol (The coniferol biosynthetic pathway is shown in sapphire. For the detailed pathway, see Supplementary Fig. 5). b Curves of cell growth and population change in the static Bgly2-Caf/Bglc2-Con coculture. c Titers of coniferol and caffeate at 48 h in the Bgly2-Caf/Bglc2-Con coculture. d Curves of cell growth and population change in the dynamic Bgly2-Caf/Bglc2-ConDmpR coculture. e Titers of coniferol and caffeate at 48 h in the Bgly2-Caf/Bglc2-ConDmpR coculture. MMCF multi-metabolite cross-feeding, IIRs the initial inoculation ratios. Genes: gdhA encodes glutamate dehydrogenase, gltBD encodes glutamate synthase, Enzymes: GdhA glutamate dehydrogenase, DmpR the caffeate-responsive transcription factor. Metabolites: PEP phosphoenolpyruvate, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

Fig. 5. Production of silybin/isosilybin using coculture engineering.

a Schematic of the three-strain coculture to accommodate the silybin/isosilybin biosynthetic pathway and convert a glycerol and glucose mixture to silybin/isosilybin (The silybin/isosilybin biosynthetic pathway is shown in blue. For the detailed pathway, see Supplementary Fig. 5). b Silybin/isosilybin titers. c Amounts of the intermediates accumulated. d Population change of the two-strain coculture. e Population change of the three-strain coculture. MMCF multi-metabolite cross-feeding. Genes: gdhA encodes glutamate dehydrogenase, gltBD encodes glutamate synthase. Enzymes: GdhA glutamate dehydrogenase, DmpR the caffeate-responsive transcription factor. Metabolites: PEP phosphoenolpyruvate, α-KG α-ketoglutarate, L-GLU L-glutamate, AAs amino acids. Data shown are mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.