Rapid production of l-DOPA by Vibrio natriegens, an emerging next-generation whole-cell catalysis chassis

Abstract

3, 4-Dihydroxyphenyl-l-alanine (l-DOPA) is a compound of high medical value and is considered effective as a treatment for Parkinson's disease. Currently, bioproduction of l-DOPA is mainly carried out by whole-cell catalysis mediated by recombinant Escherichia coli carrying heterogeneous tyrosine phenol lyase. Vibrio natriegens is increasingly attracting attention owing to its superiority, including extremely rapid growth and high soluble protein expression capacity. In this study, we attempt to develop an efficient whole-cell catalyst for l-DOPA production using V. natriegens as the chassis. The maximum soluble protein expression by V. natriegens was accomplished in 4 h at 37°C, which was equivalent to that achieved by E. coli in 16 h at 16°C. Furthermore, the maximum productivity reached over 10.0 g l^-1 h^-1 in the early stage of biocatalysis, nearly two-fold higher than previously reported. Approximately 54.0 g l^-1 l-DOPA was obtained with a catechol conversion rate greater than 95%. In conclusion, V. natriegens displays advantages, including rapid protein expression and catalytic rate in the catalysis process for l-DOPA production. These findings strongly suggest that V. natriegens has remarkable potential as a whole-cell catalysis chassis for the production of valuable chemicals.

Fig. 1

Investigation and optimization of soluble protein expression and verification of catalytic function. A. Comparison of protein expression between Vmax‐1 and BL21‐1 under different induction conditions. M, marker; C, cell lysates; S, supernatants; P, precipitates; Vmax‐1(37), Vmax‐1 induced at 37°C; BL21‐1(16), BL21‐1 induced at 16°C; BL21‐1(37), BL21‐1 induced at 37°C. B. Verification of catalytic function. C. Optimization of induction time for maximum soluble protein expression. D. Catalytic activity of whole‐cell over induction time. Black circle represented relative protein expression, red square represented OD₆₀₀. Error bars represented the SD of n = 3 biological replicates.

Fig. 2

Optimization of different parameters of catalysis system. A. Optimization of initial concentration of catechol. B. Optimization of initial concentration of sodium pyruvate. C. Optimization of initial concentration of ammonium acetate. D. The effect of different temperatures on catalytic activity. E. Stability of whole‐cell at different temperatures. Blue circle represented 20°C, orange square represented 30°C, garnet triangle represented 37°C, cyan inverted triangle represented 50°C. F. The effect of different pHs on catalytic activity. Tawny circle represented citric acid‐trisodium citrate dehydrate buffer (4.0–6.0), red square represented phosphate buffer (6.0–8.0), black triangle represented Tris‐HCl buffer (8.0–9.0), blue inverted triangle represented Gly‐NaOH buffer (9.0–10.5). Error bars represent the SD of n = 3 biological replicates.

Fig. 3

Biosynthesis of l‐DOPA by recombinant strain through whole‐cell catalysis. A. Time course of whole‐cell catalysis by Vmax‐1 without feeding. B. Biosynthesis of l‐DOPA by resting cells of Vmax‐1 with a two‐stage fed‐batch strategy. C. Biosynthesis of l‐DOPA by resting cells of Vmax‐2 with a two‐stage fed‐batch strategy. D. Biosynthesis of l‐DOPA by resting cells of BL21‐1 with a two‐stage fed‐batch strategy. Red circle represented concentration of l‐DOPA, black square represented concentration of catechol. Error bars represented the SD of n = 3 biological replicates.