Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

Abstract

BackgroundCost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production.ResultsXylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter.ConclusionsSpatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by using the promoter regions of vessel-specific genes to express xylan biosynthetic genes. The expression of xylan specifically in the xylem vessels is sufficient to complement the irx phenotype of xylan deficient mutants, while maintaining low overall amounts of xylan and lignin in the cell wall. This engineering approach has the potential to yield bioenergy crop plants that are more easily deconstructed and fermented into biofuels.

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass

“…However, the specific reoccurrence of lignin in the xylem, and not in the interfascicular fibers, of cse-2 ProVND6:CSE and cse-2 ProVND7:CSE lines resulted in cellulose-toglucose conversion efficiencies equal to those of cse-2 mutants. Similar results were obtained for a vesselspecific complementation approach of xylan mutants, where the use of 2,757-bp VND6 and 2,009-bp VND7 promoter sequences only partially recovered the irx and dwarfed phenotype of the respective xylan biosynthesis mutants (Petersen et al, 2012). Taken together, these data hint that the VND6 and VND7 promoters are not strong and/or not specific enough to fully restore the yield penalty and at the same time keep the high cellulose-to-glucose conversion efficiency of cell wall biosynthesis mutants.…”

“…Previous vessel-complementation attempts using VND6 and VND7 promoter sequences did not achieve a full restoration of vessel integrity and growth while maintaining the high sugar yield for Arabidopsis plants mutated in genes involved in lignin and hemicellulose biosynthesis (Petersen et al, 2012;Yang et al, 2013;Vargas et al, 2016). This could be the consequence of (1) the targeted cell wall biosynthesis gene or (2) the expression level and pattern conferred by the chosen promoter.…”

“…To fully restore the integrity of the vessels and the growth of ccr1 mutants, a vessel-specific promoter was required that conferred higher expression levels and/or a broader expression pattern than the previously tested VND6 and VND7 promoters (Petersen et al, 2012;Yang et al, 2013;Vargas et al, 2016). The artificial ProSNBE has been shown to direct the expression of reporter genes to xylem vessel cells of the Arabidopsis inflorescence stem (McCarthy et al, 2011).…”

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).…”

Chemically Induced Conditional Rescue of the Reduced Epidermal Fluorescence8 Mutant of Arabidopsis Reveals Rapid Restoration of Growth and Selective Turnover of Secondary Metabolite Pools

“…This association suggests that the growth of these mutants is limited by water transport deficiency resulting from these xylem abnormalities. The dwarf phenotype of ref3 and the three Arabidopsis xylan mutants irregular xylem7 (irx7), irx8, and irx9 can be effectively alleviated by expressing the cognate wild-type genes specifically in the watertransducing vessels of these mutants (Petersen et al, 2012;Yang et al, 2013). Whereas only end point phenotypic changes could be observed in these previous studies, the chemical induction system used in this work allowed us to turn on the lignin biosynthetic pathway at a certain time point and follow the early biochemical and structural changes of vasculature as Figure 9.…”