Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli

Abstract

The fundamental biology and application of bacterial exopolysaccharides is gaining increasing attention. However, current synthetic biology efforts to produce the major component of Escherichia sp. slime, colanic acid, and functional derivatives thereof have been limited. Herein, we report the overproduction of colanic acid (up to 1.32 g/L) from d-glucose in an engineered strain of Escherichia coli JM109. Furthermore, we report that chemically synthesized l-fucose analogues containing an azide motif can be metabolically incorporated into the slime layer via a heterologous fucose salvage pathway from Bacteroides sp. and used in a click reaction to attach an organic cargo to the cell surface. This molecular-engineered biopolymer has potential as a new tool for use in chemical, biological, and materials research.

Figure 1

(A) The structure of the major repeating unit in CA. l-Fucose units are highlighted in blue. (B) The wca operon in Escherichia sp. The positive regulators of CA biosynthesis, RcsA/B, are highlighted in black; gmd and fcl are highlighted in blue and pink, respectively. (C) Optimization of CA production and incorporation of non-natural sugars. AN = accession number.

Figure 2

(A) Solid-phase screening for slime production on agar plates. Slime formation results in a lustrous, shiny colony phenotype. (B) Liquid-phase screening for total carbohydrate and CA production by modified E. coli strains. CA was quantified by measuring fucose concentration in hydrolyzed samples by derivatization of extracted EPS with Cys·HCl and measuring the difference in absorbance at 427 and 396 nm. Total carbohydrate was quantified via the anthrone assay and measuring the absorbance at 620 nm. Data from quantitative experiments are presented as averages of three independent experiments to one standard deviation.

Figure 3

(A) Temperature and N-source screen to increase total carbohydrate and CA production in E. coli JM109_pRcsA. (B) CA production over time related to culture growth phase and total carbohydrate production. (C) Strain optimization to enhance CA production. CA was quantified by measuring fucose concentration in hydrolyzed samples by derivatization of extracted EPS with Cys·HCl and measuring the difference in absorbance at 427 and 396 nm. Total carbohydrate was quantified via the anthrone assay and measuring the absorbance at 620 nm. Data from quantitative experiments are presented as averages of three independent experiments to one standard deviation. *P < 0.05, **P < 0.005, ***P < 0.0005..

Figure 4

Synthesis of azide-containing fucose analogue Fuc-N₃.

Figure 5

Click functionalization of azide-modified slime. (A) Metabolic incorporation of Fuc-N₃ in engineered E. coli JM109 and CuAAc labeling of a fluorescent dye. (B) EPS production in engineered strains containing native and heterologous fucose salvage pathways when cultured in the presence of Fuc and Fuc-N₃. (C) Fluorescent read-out from click-modified EPS generated by strains engineered to metabolically incorporate Fuc-N₃. (D) Fluorescence microcopy images of FAM-labeled EPS on the surface of engineered E. coli JM109. Scale bars = 25 μM. Fucose was quantified by acid hydrolysis and addition of l-Cys, followed by spectrophotometric detection at 427 and 396 nm. Click reactions were performed using CuSO₄ (100 μM), THPTA (500 μM), 5-FAM-alkyne (50 μM), and sodium ascorbate (5 mM) in potassium phosphate buffer (pH7, 100 mM), at 30 °C and 1000 rpm. Fluorescence was measured at λ_ex = 485 nm and λ_em = 520 nm. THPTA = tris((1-hydroxy-propyl-1H-1,2,3-triazol-4-yl)methyl)amine. FAM = carboxyfluorescein. Data is presented as an average of three independent experiments to one standard deviation.