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.

Green conversion of 5‐hydroxymethylfurfural to furan‐2,5‐dicarboxylic acid by heterogeneous expression of 5‐hydroxymethylfurfural oxidase in Pseudomonas putida S12

“…As is shown in Fig. 2A, the optimal pH for P. putida_Ohec strain to produce FDCA was 8, which is similar to the results of the two previous reports (Dijkman and Fraaije, 2014; Hossain et al , 2017). Interestingly, the strain was able to retain at least 80% of its best activity within the pH range (Bao et al , 2008; Bozell and Petersen, 2010; Rosatella et al , 2011; Zhang et al , 2017) tested herein, suggesting its broad pH tolerance for practical applications.…”

“…These results compare favourably to other previous studies. The metabolically engineered R. ornithinolytica BF60 achieved an 89% conversion yield at 100 mM starting concentration of HMF (Hossain et al , 2017). Although achieved similar conversion yield as our strain in this study, the reaction time was around 1 week, which is significantly longer than the reaction time demonstrated in this study.…”

Bioengineering advancements, innovations and challenges on green synthesis of 2, 5-furan dicarboxylic acid

“…The study revealed that a high cell concentration does not enhance FDCA production due to the deficiency of oxygen in the media. This is because the pathway generates reducing power for the energy metabolism of the microorganisms which must be oxidized to sustain the flux [61]. This media engineering process is optimized for FDCA production by single parameter optimization and fractional factorial design, and production has been increased from preliminary results.…”

Enzymatic conversion reactions of 5-hydroxymethylfurfural (HMF) to bio-based 2,5-diformylfuran (DFF) and 2,5-furandicarboxylic acid (FDCA) with air: mechanisms, pathways and synthesis selectivity

“…An alternative to chemical synthesis of FDCA by harsh oxidation methods is biocatalysis using either whole-cell [15][16][17][18][19] or enzymatic [11,13,20] conversion processes. These are usually mild and environmentally friendly since they are carried out at lower temperatures and pressures while producing less toxic waste [21].…”

“…These are usually mild and environmentally friendly since they are carried out at lower temperatures and pressures while producing less toxic waste [21]. The whole-cell conversion process has proved to be efficient in production of FDCA [15][16][17][18][19] and does not require isolation of enzymes which presents an extra cost to the process. However, while these microbes produce and secrete enzymes that catalyse the oxidation of HMF to FDCA, other compounds can also be present in the reaction mixture.…”