Engineering secondary cell wall deposition in plants

Abstract

Lignocellulosic biomass was used for thousands of years as animal feed and is now considered a great sugar source for biofuels production. It is composed mostly of secondary cell walls built with polysaccharide polymers that are embedded in lignin to reinforce the cell wall structure and maintain its integrity. Lignin is the primary material responsible for biomass recalcitrance to enzymatic hydrolysis. During plant development, deep reductions of lignin cause growth defects and often correlate with the loss of vessel integrity that adversely affects water and nutrient transport in plants. The work presented here describes a new approach to decrease lignin content while preventing vessel collapse and introduces a new strategy to boost transcription factor expression in native tissues. We used synthetic biology tools in Arabidopsis to rewire the secondary cell network by changing promoter-coding sequence associations. The result was a reduction in lignin and an increase in polysaccharide depositions in fibre cells. The promoter of a key lignin gene, C4H, was replaced by the vessel-specific promoter of transcription factor VND6. This rewired lignin biosynthesis specifically for vessel formation while disconnecting C4H expression from the fibre regulatory network. Secondly, the promoter of the IRX8 gene, secondary cell wall glycosyltransferase, was used to express a new copy of the fibre transcription factor NST1, and as the IRX8 promoter is induced by NST1, this also created an artificial positive feedback loop (APFL). The combination of strategies—lignin rewiring with APFL insertion—enhances polysaccharide deposition in stems without over-lignifying them, resulting in higher sugar yields after enzymatic hydrolysis.

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

“…One option to circumvent the negative consequences of lignin down-regulation on plant performance while still achieving strong down-regulation is to specifically target the down-regulation to fibers but leaving the vessels intact. Such strategies have already proven successful in Arabidopsis thaliana (29,30).…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…Increasing cellulose synthesis and the ratio of cellulose to hemicellulose, and engineering of plants for desirable lignin content or composition by partial removal or redistribution of lignin, are obvious strategies to improve the economical feasibility of bioenergy crops [9,11,76,77]. However, there are clearly limits to such engineering, dictated by the need for optimal plant development, structural integrity, and defense [13,20]. The other limitation is resistance, in some countries, to the commercial use of genetically modified crop species.…”

“…The crystallinity and surface area accessibility of cellulose, protection of cellulose by lignin, the heterogeneous character of biomass particles, and cellulose sheathing by hemicellulose all contribute to the recalcitrance of lignocellulosic biomass to hydrolysis, and collectively represent a technical barrier to commercial deployment of cellulosic biofuel [7][8][9][10][11][12][13]. Typically, lignocellulosic biomass is preprocessed ( Figure 1A) by steam explosion, solvent treatment, or acid or alkaline treatment to reduce its recalcitrance.…”

“…The objective of such structural reengineering is to loosen the rigid cell wall structure and expose cellulose and hemicelluloses for enhanced saccharification [11,12,[16][17][18][19][20]. However, lignin genetic engineering generally results in detrimental effects on plant growth and development, and such approaches do not exclude the need for exogenous enzyme addition prior to fermentation [13,20]. There is also extensive research on the genetic engineering of microbial strains to enhance saccharification and fermentation capabilities including expression of lignocellulose digesting enzymes in existing fermentation strains (the consolidated bioprocess) [21][22][23][24][25], expanding the catabolic capacity of strains (typically by engineering C5 sugar metabolic pathways: i.e., inserting the genes encoding the enzymes of the pentose sugar degradation pathway) [26][27][28][29] and by engineering carbon flow to produce specific end-products in organisms with intrinsically broad metabolic capabilities [30].…”

Enhanced cuticle accumulation by employing MIXTA-like transcription factors

“…We hypothesized that the promoters of genes downstream of the cascade regulating cuticle formation should ideally control the expression of TF genes influencing cuticle formation. This would ensure the promoters are active at appropriate times, but would also help boost promoter activity through a positive feedback loop (Yang et al 2013). MYB106 functions during epidermal cell maturation, as indicated by the fact the trichomes of myb106 mutants remain morphologically immature (Gilding and Marks 2010;Oshima et al 2013a).…”