Integrating lignin depolymerization with microbial funneling processes using agronomically relevant feedstocks

Abstract

This work shows the integration of reductive lignin depolymerization and microbial funneling with Novosphingobium aromaticivorans to produce 2-pyrone-4,6-dicarboxylic acid (PDC) in a lignocellulosic biorefinery.

Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene

“…In this regard, a recent study of PDC production from lignin that was depolymerized by hydrogenolysis reported PDC yields from WT poplar and sorghum of 0.8% w/w and 0.6% w/w, respectively (Perez et al, 2022). Moreover, in planta production of PDC in engineered Arabidopsis showed 3% w/w accumulation in the biomass (Lin et al, 2021), which is lower than the ~5% yield that was obtained from the Arabidopsis QsuB line 2 (Figure 5).…”

“…Having established that aromatics in the APL are present as free and conjugated phenolics, we evaluated the possibility of microbially converting these aromatics to higher-value products. For this, we tested the production of PDC from APL using the engineered N. aromaticivorans PDC strain that funnels a variety of plant-derived phenolics to PDC (Perez et al, 2019;Linz et al, 2021;Perez et al, 2022).…”

“…For PDC production using N. aromaticivorans strain PDC, the APL was supplemented with glucose and ammonium sulfate, to reach final concentrations of 1 g/L each, and filtered using 0.2 µm sterile PES membrane filters. Then, 10-ml aliquots of this solution were inoculated with 2-ml aliquots of an N. aromaticivorans strain PDC culture that had been grown overnight in standard mineral base media (Perez et al, 2022) supplemented with 3 g/L glucose and incubated in an environmental growth chamber at 30 °C using a shaker at 200 rpm. The culture was incubated for 48 h; bacterial growth was monitored, and 1-ml samples were collected periodically and centrifuged at 3,300 rcf for 6 min, the supernatant was recovered and filtered, and samples were stored at −18 °C until further analysis.…”

“…QsuB transgenics have also been reported in energy crops such as poplar (Unda et al, 2022), sorghum (Lin and Eudes, 2020;Tian et al, 2022), and switchgrass (Hao et al, 2021). For microbial conversion of aromatics, we employed an engineered strain of the α-proteobacterium Novosphingobium aromaticivorans DSM 12444 (Perez et al, 2019;Perez et al, 2022) which has the capacity to funnel a variety of aromatic monomers into 2-pyrone-4,6-dicarboxylic acid (PDC), a potential polyester and adhesive precursor (Hishida et al, 2009;Shikinaka et al, 2018). We selected PDC as a target product for microbial funneling because it is an intermediate at the intersection of converging pathways for degrading different types of phenolics that can be found in deconstructed lignin (Perez et al, 2019;Perez et al, 2022).…”

“…For microbial conversion of aromatics, we employed an engineered strain of the α-proteobacterium Novosphingobium aromaticivorans DSM 12444 (Perez et al, 2019;Perez et al, 2022) which has the capacity to funnel a variety of aromatic monomers into 2-pyrone-4,6-dicarboxylic acid (PDC), a potential polyester and adhesive precursor (Hishida et al, 2009;Shikinaka et al, 2018). We selected PDC as a target product for microbial funneling because it is an intermediate at the intersection of converging pathways for degrading different types of phenolics that can be found in deconstructed lignin (Perez et al, 2019;Perez et al, 2022). phenolics from wild-type (WT) and transgenic biomass samples of Arabidopsis, poplar, and sorghum using a mild alkaline pretreatment process (see Section 4).…”