Rational engineering of Pseudomonas putida DSM 6125 for dedicated catabolic detoxification of furfural and 5-(hydroxymethyl)furfural

Abstract

The prospect of using lignocellulosic biomasses (LCBs) as cost effective feedstocks in bio-production of chemicals is hampered by the inhibitory compounds co-generated with sugars during pretreatment of LCBs. Among these inhibitory compounds, furfural and 5-(hydroxymethyl)furfural (HMF) are the most abundant, and thus, contribute the most toxicity to LCB hydrolysates. To address this challenge, we engineered a strain of Pseudomonas putida DSM 6125 to efficiently utilize furfural and HMF as carbon sources, without consuming glucose, the major sugar derived from LCB pretreatment and hydrolysis. P. putida DSM 6125 is innately incapable of utilizing xylose, the second most abundant sugar in LCB hydrolysates. To engineer a strain that selectively consumes furfural and HMF, the glucose uptake and catabolic genes namely, carbohydrate-selective porin (oprB-II), glucose dehydrogenase (gcd), and glucokinase (glk) were inactivated to obtain P. putida^{oprB-II-/gcd-/glk-} (ΔoprB-IIΔgcdΔglk). Subsequently, the furfural and HMF catabolic gene clusters of Cupriavidus basilensis DSM 11853 were chromosomally integrated into P. putida^{oprB-II-/gcd-/glk-} resulting in P. putida^Fur−HMF (ΔoprB-IIΔgcdΔglk⸬hmfFGH'Hmfs⸬hmfABCDEmfs). P. putida^Fur−HMF consumed ∼1.00–3.84 g/L furfural and 1.30–5.04 g/L HMF supplied separately as carbon sources—following adaptation to increasing inhibitor concentrations—without glucose consumption.