Laboratory Evolution Yields a Furfural-Tolerant Corynebacterium glutamicum Mutant: Exploring Its Tolerance Mechanisms and Capacity for High-Value Chemical Production

Abstract

Highly tolerant and efficient whole-cell biocatalysts for upgrading biomass-derived furfural into high-value chemicals are urgently required in biorefinery. In this study, a furfural-resistant mutant of Corynebacterium glutamicum ATCC 13032 (designated as Mutant R, MR) was developed by adaptive laboratory evolution. It grew faster than the wildtype (WT) strain in the presence of 3 g/L of furfural and was observed with an increased 2-furoic acid production from furfural. The genomic, transcriptomic, and proteomic analyses were conducted to elucidate the furfural tolerance of the MR strain. The results showed that MR resisted furfural stress by improving the ssuD expression to produce more energy (GTPs), down-regulating the DNA repair system and FudC activity to reduce energy and NADPH consumption, keeping ion homeostasis, and systematically regulating sulfur assimilation to reduce ROS damage.

The whole-cell biocatalysis results showed that MR expressing the endogenous vanillin dehydrogenase (VDH) encoded by cgl2668 identified from transcriptomic data resulted in a 47.4 % increase in 2-furoic acid production. The gene cgl2668 might be responsible for the increased 2-furoic acid production of MR. WT strain overexpressing the endogenous ADH FudC produced 6.3 g/L of furfuryl alcohol under micro-anaerobic conditions under 10.0 g/L of furfural. The exogenous alcohol dehydrogenase O16250 identified from a newly obtained furfural-tolerant strain Enterobacter hormaechei (E. H. 5.17) increased the furfural alcohol production to 7.9 g/L under micro-anaerobic conditions. Molecular docking was conducted between furfural with O16250 and FudC, and O16250 was observed with a higher affinity to furfural than FudC.

The HMF/furfural oxidoreductase CbHmfH was exogenously added to Mutant R but failed to promote furfural oxidation to FDCA and 2-furoic acid. However, the expression of SUMO fused CbHmfH with O16250 in E. coli BL21(DE3) strain completely converted 3 g/L of HMF to FDCA. Another two oxidases, GOaseM3–5 and HMFO_V367R_W466F, effective for FDCA production and obtained by paper mining, were over-expressed in Mutant R strain, still not working to oxidize HMF to FDCA. Additionally, co-expression of CbHmfH with the classic HMF acid transporter HmfT1 and its homology 4GBY_1 from E. coli didn’t promote FDCA production. The reason might be that enzymes like CbHmfH, GOaseM3–5 and HMFO_V367R_W466F might not work well in MR strain. New key enzymes of FDCA production that function normally in MR should be investigated. The developed MR and WT strains are promising hosts for aromatic acid and alcohol production.

Date of Award	12 Aug 2024
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Patrick Kwan Hon LEE (Supervisor)