Production of Salvianic Acid A from l-DOPA via Biocatalytic Cascade Reactions

Abstract

Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production.

Keywords: biocatalysis; biological engineering; l-DOPA; membrane-bound l-amino acid deaminases; molecular biology; salvianic acid A.

Figure 1

Schematic of the synthesis of salvianic acid A (SAA) from l-DOPA. DHPPA: 3,4-dihydroxyphenylalanine; ml-AAD: membrane-bound l-amino acid deaminases; d-LDH: d-lactate dehydrogenase; HPPR: hydroxyphenylpyruvate reductase; FDH: formate dehydrogenase.

Figure 2

SDS-PAGE analysis of ml-AAD expression in BL21(DE3)-pET-28a-mlaad. Lane 1, membrane fractions of BL21(DE3)-pET28a (control); lane 2, membrane fractions of BL21(DE3)-pET-28a-mlaad; The same amounts of cells were loaded in lane 1 and lane 2. Bands indicated by arrow, recombinant ml-AAD; the theoretical protein size was 51.5 kDa. Both BL21(DE3)-pET-28a-mlaad and the control were induced using 0.5 mM IPTG at 28 °C and 150 rpm for 6 h.

Figure 3

(a) Effects of pH on the relative yield of DHPPA (reactions were performed at 37 °C in reaction mixture comprised of 0.42 g·L⁻¹ recombinant cells and 20 mM l-DOPA (pH 5–10); the DHPPA yield at pH 7.5 was set as 100%); (b) effects of temperature on the relative yield of DHPPA (reactions were performed at 22–55 °C in reaction mixture comprised of 0.42 g·L⁻¹ recombinant cells and 20 mM l-DOPA (pH 7.5); the DHPPA yield at 37 °C was set as 100%); (c) effects of substrate concentration on the relative yield of DHPPA (reactions were performed at 37 °C in reaction mixture comprised of 0.42 g·L⁻¹ recombinant cells and 20-100 mM l-DOPA (pH 7.5); the DHPPA yield at 50 mM l-DOPA was set as 100%); (d) effects of cell concentration on the relative yield of DHPPA (reactions were performed at 37 °C in reaction mixture comprised of 0.11–0.84 g·L⁻¹ recombinant cells and 50 mM l-DOPA (pH 7.5); the DHPPA yield at 0.84 g·L⁻¹ cell concentration was set as 100%). Data represent the means ± SD from three independent determinations.

Figure 4

Time profile for the production of DHPPA from l-DOPA using BL21(DE3)-pET-28a-mlaad whole-cell catalysts under optimal conditions. Data represent the means ± SD from three independent determinations.

Figure 5

SDS-PAGE analysis of total cell lysates and the soluble constituents of BL21(DE3)-pETDuet-dldh-fdh and BL21(DE3)-pETDuet-hppr-fdh. Total cell lysates of BL21(DE3) were used as control (lane 1); total cell lysates and soluble constituents of BL21(DE3)-pETDuet-dldh-fdh were loaded in lanes 2 and 4, respectively; total cell lysates and soluble constituents of BL21(DE3)-pETDuet-hppr-fdh were loaded in lanes 3 and 5, respectively; bands indicated by arrows in lanes 2 and 4 were recombinant D-LDH; the theoretical protein size was 37.2 kDa; bands indicated by arrows in lanes 3 and 5 were recombinant HPPR; the theoretical protein size was 35.4 kDa. The same amounts of cells were loaded in all lanes. All cells were induced using 0.5 mM IPTG at 28 °C and 150 rpm for 6 h.

Figure 6

(a) Effects of pH on SAA production yield (the SAA yield at pH 5.5 was set as 100%). (b) Effects of temperature on SAA production yield (the SAA yield at 44 °C was set as 100%). Data represent the means ± SD from three independent determinations.

Figure 7

Effect of permeabilized BL21(DE3)-pETDuet-dldh-fdh cell concentration on SAA.