Ting-Feng Yeh

Lignin has been considered as the major barrier to enzymatic hydrolysis of the cellulose for ethanol production. This is true when lignin is chemically linked to xylan in cell walls. The long-term goal of this project is to engineer a built-in xylanolytic enzyme system specifically in wood cell walls to improve wood saccharification for ethanol production. The xylanolytic system includes a thermostable endo-â-1,4-xylanase (XynA) and a â-xylosidase (XyD) from Thermotoga maritime, the most thermophlic xylan-degradign bacterium currently known. These wood wall bound xylanolytic enzymes, which are active only at high temperatures (70-90 °C), can be used to degrade wood xylan, as a ?biological pretreatment? stage, to eliminate the lignin-xylan barrier, and to generate xylose. Cellulose in the xylan free wood should be easily saccharified by, perhaps, a mild cellulase treatment. The overall saccharification efficiency would depend on the expression level of the wall-bound xylanolytic enzymes. In this proposed project we will clone xynA and xyD genes from T. maritima MSB8. We will then quantify the expression levels in a plant background of these bacterial genes, as well as of their synthetic versions that are fully modified for optimized host codon usage. The goal of this project is to select the most appropriate sequences (native or synthetic) for high level production of the enzymes in wood. The tobacco suspension NT1 cells are the most efficient plant system for our purpose of quantifying the expression levels of the proposed sequences. The expression will be controlled by a CaMV35S promoter or a 4x CaMV35S enhancer promoter. The 4x 35S enhancer can be readily engineered to drive a xylem-specific promoter to overproduce cell wall bound xylanolytic system in wood.

We propose to investigate tree conserved miRNAs in tension wood formation. The work will help to find novel element controlling this process, and also provide new insight on cellulose biosynthesis.