A gene stacking approach leads to engineered plants with highly increased galactan levels in Arabidopsis.

Vibe M Gondolf,Rhea Stoppel,Carsten Rautengarten,Berit Ebert,Dominique Loqué,April Jm Liwanag,Henrik V Scheller

doi:10.1186/s12870-014-0344-x

Abstract

BackgroundEngineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Lignocellulosic biomass contains a high proportion of pentose residues, which are more difficult to convert into fuels than hexoses. Therefore, increasing the hexose/pentose ratio in biomass is one approach for biomass improvement. A genetic engineering approach was used to investigate whether the amount of pectic galactan can be specifically increased in cell walls of Arabidopsis fiber cells, which in turn could provide a potential source of readily fermentable galactose.ResultsFirst it was tested if overexpression of various plant UDP-glucose 4-epimerases (UGEs) could increase the availability of UDP-galactose and thereby increase the biosynthesis of galactan. Constitutive and tissue-specific expression of a poplar UGE and three Arabidopsis UGEs in Arabidopsis plants could not significantly increase the amount of cell wall bound galactose. We then investigated co-overexpression of AtUGE2 together with the β-1,4-galactan synthase GalS1. Co-overexpression of AtUGE2 and GalS1 led to over 80% increase in cell wall galactose levels in Arabidopsis stems, providing evidence that these proteins work synergistically. Furthermore, AtUGE2 and GalS1 overexpression in combination with overexpression of the NST1 master regulator for secondary cell wall biosynthesis resulted in increased thickness of fiber cell walls in addition to the high cell wall galactose levels. Immunofluorescence microscopy confirmed that the increased galactose was present as β-1,4-galactan in secondary cell walls.ConclusionsThis approach clearly indicates that simultaneous overexpression of AtUGE2 and GalS1 increases the cell wall galactose to much higher levels than can be achieved by overexpressing either one of these proteins alone. Moreover, the increased galactan content in fiber cells while improving the biomass composition had no impact on plant growth and development and hence on the overall biomass amount. Thus, we could show that the gene stacking approach described here is a promising method to engineer advanced feedstocks for biofuel production.

Highlights

Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production
Populus trichocarpa UGEc is bifunctional in vitro In order to compare the performance of different Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) for the engineering purpose, we first cloned a UGE from poplar since it might be preferable to use a poplar gene for the ultimate translation on the engineering approach to a biofuel crop such as poplar
Due to its higher sequence similarity to the non-bifunctional AtUGEs of Clade II that preferentially act in interconverting UDP-Glc to UDPGal (Figure 1), PtUGEc seemed a good candidate for generating higher amounts of galactan

Summary

Introduction

Engineering of plants with a composition of lignocellulosic biomass that is more suitable for downstream processing is of high interest for next-generation biofuel production. Plant cell walls are complex structures composed of polysaccharides influencing plant morphology, defense, growth, and signaling They constitute the most abundant biomaterial on earth and have the potential to provide a source of cheap sugars for industrial biotechnology. Two main goals of engineering plants with an altered cell wall composition in order to lower costs and improve efficiency of biofuel production is to decrease recalcitrance by decreasing the lignin content or altering the lignin composition [1,2] or to reduce the content of glucuronoxylan and at the same time increasing the content of polysaccharides composed of a larger proportion of fermentable hexoses [3]. Since β-1,4galactan is composed entirely of galactose residues, which can be fermented by yeast, an increased content of this polysaccharide would potentially improve the biomass composition for biofuel purposes. In this study we used a genetic engineering approach to increase the amount of β-1,4-galactan in stem cell walls

Methods

Results

Conclusion