Overexpression of the scopoletin biosynthetic pathway enhances lignocellulosic biomass processing

Abstract

Lignin is the main factor limiting the enzymatic conversion of lignocellulosic biomass into fermentable sugars. To reduce the recalcitrance engendered by the lignin polymer, the coumarin scopoletin was incorporated into the lignin polymer through the simultaneous expression of FERULOYL-CoA 6'-HYDROXYLASE 1 (F6'H1) and COUMARIN SYNTHASE (COSY) in lignifying cells in Arabidopsis. The transgenic lines overproduced scopoletin and incorporated it into the lignin polymer, without adversely affecting plant growth. About 3.3% of the lignin units in the transgenic lines were derived from scopoletin, thereby exceeding the levels of the traditional p-hydroxyphenyl units. Saccharification efficiency of alkali-pretreated scopoletin-overproducing lines was 40% higher than for wild type.

Fig. 1.. Biosynthesis and incorporation of scopoletin into the lignin polymer.

Feruloyl-CoA is biosynthesized via the phenylpropanoid pathway. F6′H1, FERULOYL-CoA 6′-HYDROXYLASE 1; COSY, COUMARIN SYNTHASE. It is hypothesized that, because of the presence of a conjugated carbonyl in the scopoletin monomer, the 8-O-4 bond between scopoletin and the A unit in the engineered lignin polymer becomes easier to cleave under alkaline conditions whether or not the 8-position is substituted. The red arrow indicates the bond with increased reactivity. Note that alkaline conditions will also hydrolyze the cyclic ester (lactone), thereby cleaving the bond indicated with an asterisk (*) and resulting in increased hydrophilicity and thus in a polymer that is easier to extract into aqueous solvents. Note that because scopoletin is shown as a lignin monomer, its numbering follows standard lignin conventions rather than formal coumarin numbering.

Fig. 2.. Phenolic profiling of SCOP lines and WT.

(A) Average ion intensities of scopoletin and its glucoside scopolin. For each compound, the average ion intensity was significantly higher in SCOP lines as compared to WT (P < 0.001, ANOVA followed by post hoc Tukey test; n = 9; error bars represent the SE). (B) Principal components analysis of the phenolic profile of SCOP-1, SCOP-2, and WT lines. PC, principal component. Venn diagrams represent the 95% confidence regions of all detected m/z features. (C) Overview of all (partially) characterized differential metabolites.

Fig. 3.. Bright-field and fluorescence microscopy of SCOP lines.

(Top) Bright-field microscopy revealed a yellow color in SCOP lines. (Middle) Phloroglucinol-HCl (Wiesner)–stained inflorescence stem sections were visualized using bright-field microscopy. (Bottom) Autofluorescence of inflorescence stem sections upon excitation at 405 nm. V, vessel; XF, xylary fiber; IF, interfascicular fiber; P, phloem. Scale bars, 100 μm. Representative for n = 3 biological replicates.

Fig. 4.. NMR spectrum of the lignin aromatic region.

Partial short-range ¹H–¹³C HSQC NMR spectra from isolated ELs from cell walls of the main stem of senesced Arabidopsis plants. WT (A) and SCOP (B and C) lines were analyzed. The colors of the contours correspond to the structures drawn. H peaks are indicated as trace amounts as their signals coincide with those of Phe_3/5. In the difference spectrum (D), hot-colored contours represent positive, whereas cold (light cyan) contours represent negative differences after subtracting (A) from (B); the scopoletin peaks colored in solid magenta were also positive. A scopoletin standard (E) was used to identify resonance peaks related to scopoletin incorporation in the difference spectrum. ppm, parts per million (Hz/MHz).

Fig. 5.. Incorporation of scopoletin into the lignin polymer.

GC-MS analysis of selected DFRC cleavage products from isolated EL of (A) WT, (B) SCOP-1, and (C) SCOP-2 lines. 4-O etherified scopoletin and 8-O etherified scopoletin were detected with an m/z of 193 and 192, respectively. (D) Putative routes of scopoletin incorporation together with the expected chemical markers after DFRC analysis. Scopoletin covalently couples into the lignin polymer via radical coupling, giving rise to scopoletin as a starting unit (route i), as an internalized unit (route ii), and as a phenolic end unit (route iii). Ar stands for aryl, e.g., a G or S unit, or a unit derived from a second scopoletin monomer. M_w, weight-average molecular weight.

Fig. 6.. Enzymatic cellulose-to-glucose conversion of SCOP lines.

Before the saccharification, stem segments were pretreated with 62.5 mM NaOH at 90°C for 3 hours. (A) Released glucose was expressed as a percentage of the total cellulose in the respective lines. Saccharification was ended after 72 hours. At each time point, cellulose-to-glucose yield was significantly higher in both SCOP lines as compared to that of WT. Significance levels are indicated by ^● and * for SCOP-1 and SCOP-2 lines, respectively, compared to WT [* and ^●, 0.05 > P > 0.01; ** and ^●●, 0.01 > P > 0.001; *** and ^●●●, P < 0.001 (ANOVA followed by post hoc Tukey test; n = 10)]. (B) Biomass of SCOP lines and controls after 62.5 mM NaOH pretreatment and 72 hours of enzymatic hydrolysis. SCOP stem pieces are structurally degraded, whereas those of WT remain largely intact. Scale bars, 4 mm.