Metabolic engineering and optimization of Escherichia coli co-culture for the de novo synthesis of genkwanin

Abstract

Genkwanin has various significant roles in nutrition, biomedicine, and pharmaceutical biology. Previously, this compound was chiefly produced by plant-originated extraction or chemical synthesis. However, due to increasing concern and demand for safe food and environmental issues, the biotechnological production of genkwanin and other bioactive compounds based on safe, cheap, and renewable substrates has gained much interest. This paper described recombinant Escherichia coli-based co-culture engineering that was reconstructed for the de novo production of genkwanin from d-glucose. The artificial genkwanin biosynthetic chain was divided into 2 modules in which the upstream strain contained the genes for synthesizing p-coumaric acid from d-glucose, and the downstream module contained a gene cluster that produced the precursor apigenin and the final product, genkwanin. The Box-Behnken design, a response surface methodology, was used to empirically model the production of genkwanin and optimize its productivity. As a result, the application of the designed co-culture improved the genkwanin production by 48.8 ± 1.3 mg/L or 1.7-fold compared to the monoculture. In addition, the scale-up of genkwanin bioproduction by a bioreactor resulted in 68.5 ± 1.9 mg/L at a 48 hr time point. The combination of metabolic engineering and fermentation technology was therefore a very efficient and applicable approach to enhance the production of other bioactive compounds.

Keywords: De novo production; Escherichia coli; Co-culture; Genkwanin; Metabolic engineering.

Graphical Abstract

Engineering E. coli co-culture for production of genkwanin.

Fig. 1

De novo genkwanin production using the monoculture Escherichia coli R1E in the minimal M9 and complex MY1 media. The minimal M9 medium was exogenously fed with 20 g/L glucose, 2 g/L malonate, and 2 g/L methionine. The complex MY1 medium was exogenously supplemented with 10 g/L glucose, 2 g/L malonate, and 2 g/L methionine. (a) Comparison of growth profile in the MY1 plus (OD_MY1) and in the M9 plus (OD_M9) by indicating OD₆₀₀ values. (b). Comparison of genkwanin production in the MY1 plus (GEN_MY1) and in the minimal M9 plus (GEN_M9). The experiments were conducted with an initial net cell density of 3.5 × 10⁶ cells per mL of culture. The error bars represent the standard deviation of the experimental measurements for at least three independent experiments. Two-tailed Student’s t-tests were performed to determine the statistical significance of two group comparisons, α = 0.05.

Fig. 2

Schematic representation of Escherichia coli modular coculture system for production of genkwanin starting from d-Glucose. The upstream strain was a p-coumaric acid over-producer, and the downstream strain was specialized in converting p-coumaric acid to genkwanin. G6-P, glucose-6-phosphate; PYR, pyruvate; F6P, fructose-6-phosphate; G3P, glucose 3-phosphate; PpsA, phosphoenolpyruvate synthase; TktA, transketolase A; PEP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; E4P, erythrose 4-phosphate; AroG, DAHP synthase; DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; SHIK, shikimate 5-phosphate; S3P, 3-enolpyruvylshimate-5-phosphate; PPA, phenylpyruvate; PPY, phenylpyruvate, L-Phe, L-phenylalanine; FjTAL, tyrosine ammonia-lyase; tyrB, tyrosine aminotransferase ammonia-lyase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; matC, malonate transporter protein; matB, malonyl-CoA synthetase; pCA, p-coumaric acid; FNSI, flavone synthase I; and F1OMT, flavonoid 7-O-methyltransferase.

Fig. 3

Investigating the biosynthetic performance of p-coumaric acid by the monoculture Escherichia coli MCA and R1 in the MY1 medium plus 20 g/L d-glucose. (a) Growth profile of E. coli MCA (OD_MCA) and R1 strain (OD_R1). (b) Comparison of the p-coumaric acid production by MCA (pCA_MCA) and R1 (pCA_R1). The R1 was reconstructed based on MCA. The experiments were conducted with an initial net cell density of 3.5 × 10⁶ cells per mL of culture. (c) The difference between metabolically engineered E. coli MCA and E. coli R1. The error bars represent the standard deviation of the experimental measurements for at least three independent experiments.

Fig. 4

Production of genkwanin by the monoculture Escherichia coli F1, F2, and F3 strains in MY1 medium plus 10 g/L glucose, 2 g/L malonate, and 2 g/L methionine via the whole-cell bioconversion using the substrate p-coumaric acid. (a) Growth profile of F1, F2 and F3 strains (OD_F1, OD_F2, and OD_F3). (b) Comparison of genkwanin production by those strains (GEN_F1, GEN_F2, and GEN_F3). F1, F2, and F3 were reconstructed based on API strain to improve genkwanin production. The experiments were conducted with an initial net cell density of 3.5 × 10⁶ cells per mL of culture. The error bars represent the standard deviation of the experimental measurements for at least three independent experiments.

Fig. 5

Optimization of the genkwanin production by the co-culture using RSM-BBD. (a–f) Response surface plots for genkwanin production as a function of the four variables under study: inoculation ratio (% R1), inducer (IPTG) concentration (mM), induction time (H), and temperature (°C). These plots were obtained for a given pair of variables while maintaining the other one fixed at its zero value.

Fig. 6

Genkwanin bioproduction by the Escherichia coli R1:F3 co-culture using a fed-batch bioreactor. Initial glucose, malonate, and methionine concentrations were 5, 1, and 1 g/L, respectively. Those nutrients were then gradually supplied at 6, 12, and 18 h to reach final concentrations of 20, 4, and 4 g/L, respectively. Each constituent strain (R1 and F3) was used with the initial net cell density of 4.5 × 10⁶ cells per mL of each culture. The error bars represent the standard deviation of the experimental measurements for at least three independent experiments.