Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by Pseudomonas putida KT2440

Abstract

The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

Bio-upcycling of polyethylene terephthalate

“…The use of enzyme cocktails will also enable feedstock flexibility, especially when combined with microbes engineered to accept other plastic-derived substrates. For example, the novel PET-like polymer polyethylene furanoate (PEF) is also degraded by a PET hydrolase 17 , and P. putida can be engineered to degrade the resulting 2,5-furandicarboxylic acid 57,58 . It is worth noting that logistics is a major hurdle in lignocellulosic biotech since often completely new infrastructure has to be built (i.e., from forest to factory).…”

Challenges in lipid production from lignocellulosic biomass using Rhodosporidium sp.; A look at the role of lignocellulosic inhibitors

“…In general, these reactions constitute the beginning of the degradation of compounds such as furfural and 5‐HMF through biological pathways . Nevertheless, these microorganisms use compounds necessary for the production of energy in the cell (NADH or NADPH) to transform furfural and 5‐HMF (furfuryl alcohol and 5‐hydroxymethyl furfuryl alcohol, respectively) and to perform the different reactions involved in the synthesis of the lipids . On the other hand, this type of inhibitor removal has two major disadvantages.…”

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

“…With an amount of 2.5 g/L, the maximum OD 600 increased from 12.4 to 14.6. Since P. putida KT2440 wild type is not able to use furfural and HMF as substrates for growth, Guarnieri et al () engineered a strain via genomic integration of the hmf gene cluster. Consequently, the strain metabolized HMF and furfural via the intermediate 2‐furoic acid.…”

“…This is consistent with results obtained in a study in which a lag phase of 24 hr was observed during the cultivation of P. putida KT2440 with lignocellulose hydrolyzates supplemented with 2 g/L HMF and 1 g/L furfural. An explanation for this is the metabolization of furfural aldehydes to less toxic dead-end alcohol counterparts (furfuryl alcohol and HMF furfuryl alcohol) practiced by many microorganisms, which has been reported in the past (Guarnieri, Ann Franden, Johnson, & Beckham, 2017). Furthermore, in theory, part of the highly reactive furfural and HMF could have formed less toxic macromolecules over time of the cultivation.…”