Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method

Abstract

D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered Escherichia coli strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL^-1 respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification.

Keywords: D-DIBOA; nitroreductase NfsB; spectrophotometric method; whole-cell biocatalysis.

Figure 1

(a) DIBOA and D-DIBOA molecular structures and (b) D-DIBOA synthesis which involves two steps: The first step is a chemical reaction that using 2-nitrophenol starting material and ethyl bromoacetate to produce ethyl-2-(2′-nitrophenoxi)acetate (the precursor in the second step) by a nucleophilic substitution. The second step involves the reduction of a nitro group followed by a cyclization that is catalyzed by the nitroreductase (NfsB) enzyme (dashed box). This second reaction is catalyzed by NfsB nitroreductase which is NAD(P)H-dependent (*).

Figure 2

Molecular structure of the complex Fe (III)-(D-DIBOA)₃.

Figure 3

Spectral absorbance determination of Fe (III)-(D-DIBOA)₃ complex, specificity, and optimization of calibration curves. (a) Spectral absorbance of Fe(III)-(D-DIBOA)₃ complex in ethanol and M9 medium; (b) spectral absorbance curves of: D-DIBOA (DD), precursor (PC), 2-nitrophenol (NP) dissolved in ethanol (Et), in the presence of FeCl₃ also dissolved in Et; (c) spectral absorbance curves of: D-DIBOA, precursor, 2-nitrophenol dissolved in M9, in the presence of FeCl₃ dissolved in water; (d) calibration curves of D-DIBOA (mM) by adding FeCl₃ 0.2 and 0.3 M with pH < 1 and measured spectrophotometrically at a wavelength of 570 nm in a microplate reader.

Figure 4

Correlation of OD₅₇₀ using the D-DIBOA spectrophotometric method developed in this work versus the HPLC analysis (peak areas) in the method previously established (standards 0.25–4 mM).

Figure 5

Plots of biotransformation experiments for the optimization of D-DIBOA production. The left panels show D-DIBOA concentrations (mM) quantified by the spectrophotometric method (black circles) and precursor concentrations calculated stoichiometrically (white circles). In the right panels are shown plots of biomass (cross) and glucose concentrations (triangles). (a) Biotransformation assays keeping the precursor concentration at 2.2 mM by adding a fresh load every hour (from 2 to 10 h) with no glucose addition; (b) biotransformations with the same precursor loading strategy but keeping the glucose concentration at 22.2 mM every hour (from 2 to 10 h); (c) biotransformation assays keeping the precursor concentration at 2.2 mM by adding a fresh load every hour from 2 to 7 h and a single glucose pulse at 7 h to reach 22.2 mM.

Figure 6

Tolerance test on BW25113/pBAD-NfsB (black bars) and ΔlapAΔfliQ/pBAD-NfsB (grey bars) Escherichia coli strains for increasing D-DIBOA concentrations. Bar charts show relativized values of bacterial biomass grown for 12 h with respect the biomass at the moment in which D-DIBOA was added, in an OD_600nm = 0.6. Bacterial growth was measured in absence (positive control, C; and positive control with MeOH, C´) or in the presence of different concentrations of D-DIBOA. The error bars represent the standard deviations of three independent replicates.