High-yield production of lignin-degrading enzymes for biocatalysis of renewable chemicals and biofuel

Researchers in Prof. Elizabeth Sattely's laboratory have developed a high-yield, scalable plant-based protein expression system to produce lignin-degrading enzymes for converting waste lignin into useful carbon-based platform chemicals. Lignin is the second most abundant biopolymer, but is underutilized as a renewable source of commodity chemicals because it is difficult to break down chemically. Certain fungal enzymes can be used as biocatalysts to degrade lignin, but traditional expression systems (e.g., in yeast) cannot produce sufficient quantities of those enzymes in a reliable, scalable manner.

This technology solves the enzyme-production problem by engineering tobacco plants (N. benthamiana) with an inducible Agrobacterium-mediated platform. These transgenic plants can produce significant quantities of a large panel of soluble ligninases. A crude leaf extract can then be used to degrade lignin in vitro. The basic platform can be expanded to produce other enzymes of interest, providing a robust, reliable, versatile system to produce biocatalysts. This system could capitalize on the massive lignin waste stream to produce biofuels and industrial chemicals.

Stage of Research
The inventors have demonstrated that multiple enzymes from major fungal lignin-modifying enzyme families can be produced at high titers using with this tobacco expression system. Half of all ligninases tested from 4 major families were successfully expressed. Furthermore, the inventors used the tobacco-produced enzymes to catalyze cleavage of a model lignin dimer in vitro.

Overview of heterologous production of lignin-degrading enzymes in N. benthamiana. Production of enzymes in N. benthamiana was driven by a 35S promoter on a pEAQ expression vector and exported to the plant apoplast via fusion to the signal peptide of dirigent protein from Sinopodophyllum hexandrum (PhDIR SP). The pEAQ vector includes 5' and 3' untranslated regions from cowpea mosaic virus (5' CPMV and 3' CPMV) to enhance expression levels. Hemaaglutinin (HA), c-myc (myc), and hexahistidine (his6) affinity tags were included as N- and/or C-terminal fusions where indicated.