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

Abstract

The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cell-wall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta. One such genetic modification is the expression of the bacterial quinate and shikimate utilization B (qsuB) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans. Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (∼5% w/w), followed by QsuB sorghum (∼1.1% w/w), and QsuB poplar (∼0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.

Engineering Novosphingobium aromaticivorans to produce cis,cis -muconic acid from biomass aromatics

“…All the PCA and 4-HBA were transformed during incubation, with only trace amounts of vanillic acid remaining after the incubation. Umana et al demonstrated that the glycosylated forms of PCA and vanillic acid are degraded by N. aromaticivorans , and as PCA is an intermediate in the degradation of vanillic acid and 4-HBA, this observation leads us to conclude that these strains were producing cc MA from all the aromatics quantified in the biomass APL ( 51 ). However, the greater than 100% cc MA yield in the NaDec_ cc MA and EcDec_ cc MA cultures grown in the presence of APL suggests that both of these engineered strains are capable of metabolizing other unidentified aromatics present in the APL.…”

“…It is known that transgenic plants expressing the quinate and shikimate utilization B ( qsuB ) gene increase the accumulation of aromatics, notably PCA, found in biomass ( 50 ). Previously, we have shown that these QsuB transgenic poplar plants can be used as a source of aromatics for the conversion of biomass aromatics to PDC ( 51 ). Thus, we tested the ability of the NaDec_ cc MA and EcDec_ cc MA strains to produce cc MA from aromatics derived from a transgenic poplar QsuB plant.…”

“…An aqueous solution containing both phenolic monomers and glycosylated forms of PCA and vanillic acid was obtained from QsuB poplar biomass using a mild alkaline pretreatment that cleaves ester linkages (Fig. S9) ( 51 ). After acid treatment of the APL to release the glycosylated phenolic compounds ( 51 ), the major identifiable phenolics in this material were PCA, vanillic acid, and 4-HBA with trace amounts of ferulic acid and 4-coumaric acid (Table S11).…”

“…After acid treatment of the APL to release the glycosylated phenolic compounds ( 51 ), the major identifiable phenolics in this material were PCA, vanillic acid, and 4-HBA with trace amounts of ferulic acid and 4-coumaric acid (Table S11). It is known that N. aromaticivorans is able to metabolize both free and glycosylated phenolics ( 51 ), so the concentration of known phenolics derived from the Qsub poplar biomass was calculated to include the concentration of the glycosylated phenolics ( 51 ). This calculation showed that we obtained 0.42 ± 0.03 mM of total aromatics in the Qsub APL with PCA accounting approximately 60% of the total aromatics ( Fig.…”

“…Furthermore, since the mild alkaline pretreatment process used to generate the APL cleaves the ester bonds and liberates soluble metabolites without breaking down the lignin backbone ( 51 ), it is likely that these unidentified aromatics are not oligomeric forms of deconstructed lignin. Nevertheless, the diversity of aromatic catabolic pathways available in N. aromaticivorans could be a valuable characteristic of N. aromaticivorans since lignocellulosic biomass deconstruction methods that are more aggressive than the mild alkaline pretreatment used in this study would be expected to produce a range of aromatic monomers, dimers, and oligomers, as well as sugar conjugates and other organic materials that could be used to support growth of engineered strains.…”