Reducing biomass recalcitrance by heterologous expression of a bacterial peroxidase in tobacco (Nicotiana benthamiana)

Abstract

Commercial scale production of biofuels from lignocellulosic feed stocks has been hampered by the resistance of plant cell walls to enzymatic conversion, primarily owing to lignin. This study investigated whether DypB, the lignin-degrading peroxidase from Rodococcus jostii, depolymerizes lignin and reduces recalcitrance in transgenic tobacco (Nicotiana benthamiana). The protein was targeted to the cytosol or the ER using ER-targeting and retention signal peptides. For each construct, five independent transgenic lines were characterized phenotypically and genotypically. Our findings reveal that expression of DypB in the cytosol and ER does not affect plant development. ER-targeting increased protein accumulation, and extracts from transgenic leaves showed higher activity on classic peroxidase substrates than the control. Intriguingly, in situ DypB activation and subsequent saccharification released nearly 200% more fermentable sugars from transgenic lines than controls, which were not explained by variation in initial structural and non-structural carbohydrates and lignin content. Pyrolysis-GC-MS analysis showed more reduction in the level of lignin associated pyrolysates in the transgenic lines than the control primarily when the enzyme is activated prior to pyrolysis, consistent with increased lignin degradation and improved saccharification. The findings reveal for the first time that accumulation and in situ activation of a peroxidase improves biomass digestibility.

The Maize Corngrass1 miRNA-Regulated Developmental Alterations Are Restored by a Bacterial ADP-Glucose Pyrophosphorylase in Transgenic Tobacco

“…Leaf explants (~0.5 mm 2 ) of 4–6-week-old tobacco were infiltrated with Agrobacterium harboring the expression vectors pPZP- NPTII - Cg1 , pPZP- NPTII - glgC , and pPZP- NPTII - Cg1-glgC or the empty vector pPZP-NPTII for five minutes in the presence of 200 μ M acetosyringone. Handling of transformed tissues, selection and regeneration, and maintenance of transgenic lines were performed based on Ligaba-Osena et al [ 39 ].…”

“…Expression of transgenes ( Cg1 and glgC ) and putative Cg1 -targets was studied by quantitative real-time RT-PCR (qPCR) as described previously [ 39 ]. Primers used for gene expression analysis are listed in Table S1 ; 18S RNA was used as an internal control.…”

“…Sugars in the hydrolysate obtained after biomass saccharification were characterized filtered (0.22 μ m) for quantifying sugars using Dionex Ion Exchange Chromatography 3000 equipped with an electrochemical detector (Dionex, Sunnyvale, USA) as previously described [ 39 ]. The sugar concentration from the IC reading was converted to milligrams of sugar per gram of dry matter or milligrams of sugar per plant.…”

Bacterial enzymes for lignin depolymerisation: new biocatalysts for generation of renewable chemicals from biomass

“…In model studies, oxidative cleavage by Dyp-type peroxidases has only been observed using units containing a free phenolic 4-hydroxyl group [6], therefore it seems likely that for breakdown of polymeric lignin breakdown, they cleave from the ends of a lignin chain (exo-cleavage), rather than in the middle of a chain (endo-cleavage). Expression of R. jostii DypB in tobacco plants has been shown to yield 200% more fermentable sugars, and reduced lignin content, demonstrating that Dyp-type peroxidases can be expressed heterologously to depolymerise lignin [39].…”

Characteristics of calcium lignin from pulping waste liquor and application for the treatment middle-stage wastewater of paper making

“…Therefore, utilization of renewable materials becomes an encouraging way to resolve the problem to maintain the sustainable development of human beings [2]. As the second-most-abundant biomass resource on Earth, after cellulose, lignin is a natural renewable resource with great potential [3,4]. Its chemical structure is complex [5] and its chemical composition varies with irregular arrays, resulting in valuable physicochemical properties [6,7].…”