Combinatorial synthetic pathway fine‐tuning and comparative transcriptomics for metabolic engineering of Raoultella ornithinolytica BF60 to efficiently synthesize 2,5‐furandicarboxylic acid

Abstract

The compound 5-hydroxymethylfurfural (HMF) has attracted much attention due to its versatility as an important bio-based platform chemical. Here, we engineered Raoultella ornithinolytica BF60 as a whole-cell biocatalyst for a highly efficient synthesis of 2,5-furandicarboxylic acid (FDCA) from HMF. Specifically, various expression cassettes of key genes, such as hmfH (gene encoding HMF/furfural oxidoreductase [HmfH]) and hmfo (gene encoding HMF oxidase), were designed and constructed for fine-tuning FDCA synthesis from HMF. The FDCA titer reached 108.9 mM with a yield of 73% when 150 mM HMF was used as the substrate. This yield was 16% higher than that without balancing key gene expression in FDCA synthetic pathways. Additionally, to strengthen HmfH expression at the translational level, ribosomal binding site (RBS) sequences, which were computationally designed using the RBS calculator, were assembled into HmfH expression cassettes. The HmfH expression in the presence of these sequences enhanced FDCA titer to 139.6 mM with a yield of 93%. Next, previously unknown candidate genes, such as aldR, dkgA, akR, AdhP1, and AdhP2, which encode enzymes that catalyze the reactions leading to the formation of the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol) from HMF, were identified by RNA-sequencing-based transcriptomics. Combinatorial deletion of these five candidate genes led to an 88% reduction in HMF alcohol formation and 12% enhancement in FDCA production (175.6 mM). Finally, FDCA synthesis was further improved by the substrate pulse-feeding strategy, and 221.5 mM FDCA with an 88.6% yield was obtained. The combinatorial synthetic pathway fine-tuning and comparative transcriptomics approach may be useful for improving the biocatalysis efficiency of other industrially useful compounds.

Redox-Switchable Biocatalyst for Controllable Oxidation or Reduction of 5-Hydroxymethylfurfural into High-Value Derivatives

“…These results suggest that HMF might be first converted to HMF alcohol, then re-oxidized to HMF at a harmless lower concentration, and subsequently oxidized to its acid forms. The HMF biodegradation pattern by P. azotifigens F18 was similar to that of Raoultella ornithinolytica BF60 as reported before 22 but with less accumulation of FDCA. Similar phenomena have also been confirmed in the common biodetoxification mechanism of microorganisms toward furan.…”

“…The reasons might be that HMFCA can also be oxidized by HMF oxidase (HMFO) via route B or other endogenous nonspecific dehydrogenases in route A ( Scheme 1 ). 22 Therefore, the HMFO gene that is responsible for the oxidation of HMFCA was further deleted to verify its functions. The gene manipulation process and the verification experiment are shown in the Supporting Information .…”

“…The gene encoding HMF oxidase ( hmfO ) was further mutated to prevent the conversion of HMF via route B ( Scheme 1 ). 22 The native sequences of the selected genes cloned from P. azotifigens F18 are shown in the Supporting Information . The gene deletion was performed using the λ-Red recombination system.…”

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

“…This led to enhancement (12%) in FDCA production of 175.6 mM. This FDCA synthesis is again improved by-fed batch strategy (50,25,25,25, and 25 mM HMF in every 12 h from 24-72 h) and 221.5 mM FDCA produced with 88.6% yield from 250 mM HMF [73]. Even though the FDCA amount is approximate to 34.5 g/L (221.5 mM) its conversion rate (HMF to FDCA) is less as compared to other metabolically engineered strains.…”

Widespread distribution of hmf genes in Proteobacteria reveals key enzymes for 5-hydroxymethylfurfural conversion

“…An enzyme playing a crucial role in the upper pathway of furans biodegradation is the HMF oxidoreductase encoded by hmfH , previously described in C. basilensis HMF14, which has been extensively studied by its ability to convert HMF into FDCA in heterologous strains P. putida S12 and Raoultella ornithinolytica BF60, turning a valuable tool for production of this high-value precursor [22] , [26] , [50] , [51] , [52] . HmfH is a member of the glucose-methanol-choline (GMC) oxidoreductase family and mainly converts HMFCA into FFCA, and secondarily it would be also involved in the subsequent oxidation into FDCA [18] , [26] .…”