Metabolic Engineering of Raoultella ornithinolytica BF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural

Abstract

2,5-Furandicarboxylic acid (FDCA) is an important renewable biotechnological building block because it serves as an environmentally friendly substitute for terephthalic acid in the production of polyesters. Currently, FDCA is produced mainly via chemical oxidation, which can cause severe environmental pollution. In this study, we developed an environmentally friendly process for the production of FDCA from 5-hydroxymethyl furfural (5-HMF) using a newly isolated strain, Raoultella ornithinolytica BF60. First, R. ornithinolytica BF60 was identified by screening and was isolated. Its maximal FDCA titer was 7.9 g/liter, and the maximal molar conversion ratio of 5-HMF to FDCA was 51.0% (mol/mol) under optimal conditions (100 mM 5-HMF, 45 g/liter whole-cell biocatalyst, 30°C, and 50 mM phosphate buffer [pH 8.0]). Next, dcaD, encoding dicarboxylic acid decarboxylase, was mutated to block FDCA degradation to furoic acid, thus increasing FDCA production to 9.2 g/liter. Subsequently, aldR, encoding aldehyde reductase, was mutated to prevent the catabolism of 5-HMF to HMF alcohol, further increasing the FDCA titer, to 11.3 g/liter. Finally, the gene encoding aldehyde dehydrogenase 1 was overexpressed. The FDCA titer increased to 13.9 g/liter, 1.7 times that of the wild-type strain, and the molar conversion ratio increased to 89.0%.

Importance: In this work, we developed an ecofriendly bioprocess for green production of FDCA in engineered R. ornithinolytica This report provides a starting point for further metabolic engineering aimed at a process for industrial production of FDCA using R. ornithinolytica.

Keywords: 2,5-furandicarboxylic acid; 5-hydroxymethyl furfural; Raoultella ornithinolytica BF60; metabolic engineering; whole-cell biocatalyst.

FIG 1

Metabolic pathway of 5-HMF and FDCA in R. ornithinolytica BF60. CoA, coenzyme A.

FIG 2

LC-MS analysis and UPLC-ESI-TOF-MS analysis of FDCA produced by R. ornithinolytica BF60 strains. (A) Selected-ion chromatogram analysis of FDCA (m/z 155.0 [M − H]⁻) produced by the authentic FDCA standard. (B) Selected-ion chromatogram analysis of FDCA (m/z 155.0 [M − H]⁻) produced by R. ornithinolytica BF60. (C) UPLC trace for authentic standard of FDCA (Rt = 4.65). (D) UPLC trace for FDCA produced by R. ornithinolytica BF60 (Rt = 4.61). Rt, retention time.

FIG 3

Influence of pH, temperature, substrate concentration, and cell concentration on whole-cell catalytic activity. (A) Influence of pH on the production rate. (B) Influence of temperature on the production rate. (C) Influence of substrate concentration on the production rate. (D) Effects of the biocatalyst concentration on the production rate. The data shown represent the averages ± standard deviations (SD) of results of three independent replicates.

FIG 4

Activity of FDCA decarboxylase (gray bar) and 5-HMF reductase (white bar) in the wild-type strain, the dicarboxylic acid decarboxylase mutant (RTBF60-1), and the dicarboxylic acid decarboxylase and aldehyde reductase mutant (RTBF60-2). The data shown represent the averages ± SD of results of three independent replicates.

FIG 5

Effect of mutagenesis on the molar conversion efficiency and time profile for product formation. (A) Efficiency of conversion of different mutant and overexpressed strains from 5-HMF to FDCA production (mol/mol). (B) Time profile for the production of FDCA from 100 mM 5-HMF by different whole-cell biocatalysts. Wild type, ○; RTBF60-1, ■; RTBF60-2, Δ; RTBF60-3, •. Data were analyzed using the Student t test. P values of less than 0.05 were considered statistically significant (the P value for comparisons of the wild type to the RTBF60-1 strain was 0.0004; the P value for comparisons of the wild type to the RTBF60-2 strain was 0.0002; and the P value for comparisons of the wild type to the RTBF60-3 strain was 0.0004). The data shown represent the averages ± SD of results of three independent replicates.