Oral Presentation 2-03

Genetic System Development, Metabolic Engineering, Bioenergetics and Kinetics Relevant to Ethanol Production Using Thermophilic Bacteria

Lee R. Lynd,^1,2 Sunil Desai,¹ Mikhail Tyurin,¹ Yiheng Zhang,¹ Yanpin Liu,¹ A. Joe Shaw¹ and Jonathan Mielenz¹

¹Thayer School of Engineering

Dartmouth College

Hanover, NH 03755

Phone: (603)646-2231

Fax: (603)646-2277

E-mail: lee.lynd@dartmouth.edu

²Department of Biological Sciences

Fermentation of pretreated cellulosic biomass to a product of interest without added saccharolytic enzymes is a potential means to dramatically lower the cost of producing ethanol and other commodity fermentation products. Thermophilic anaerobic bacteria are attractive starting points for organism development pursuant to such consolidated bioprocessing (CBP) in light of their collective ability to rapidly ferment cellulose, xylose, and other biomass components. Production of organic acids (acetic and lactic) is a key limitation of available thermophilic strains for ethanol production because it lowers the product yield and also because the ethanol titer appears to be limited by salt accumulation resulting from organic acid neutralization (Lynd et al. 2001. Biotech. Prog. 17:118-125). Studies pursuant to elimination of organic acid production in thermophilic bacteria have been impeded by the absence of techniques for high-frequency gene transfer. In addition, concern has been expressed about the bioenergetic feasibility of consolidated bioprocessing in light of the low ATP yield of anaerobic fermentation together with the high ATP requirement for cellulase synthesis.

We report here development of high-frequency (> 10⁵ transformants/µg DNA) gene transfer systems for the cellulose-utilizing Clostridium thermocellum (strains DSM 1313 and ATCC 27405) and the xylose-utilizing Thermoanaerobacterium saccharolyticum (strain YS485). Results will also be presented for transformation of a second xylose-utilizing thermophile, Thermoanaerobacterium thermosaccharolyticum (strain HG8). Development and characterization of knock-out mutants deficient in enzymes associated with organic acid formation, obtained using our new transformation protocols, will be described. Data will be presented that support the following points relative to the bioenergetics and kinetics of cellulose utilization by C. thermocellum: 1) cellodextrins of length approximately 4 rather then cellobiose are the primary hydrolysis product taken up by cells growing on cellulose, implying that the primary enzyme activity responsible for cellulose solubilization is not cellobiohydrolase as in aerobic fungal systems; 2) once in the cell, cellodextrins are cleaved phosphorolytically by cellodextrin (and cellobiose) phosphorylase rather than hydrolytically; 3) bioenergetic benefits specific to growth on cellulose are several-fold larger than the bioenergetic cost of cellulase synthesis; 4) the C. thermocellum cellulosome appears to be several-fold more active when part of a ternary cellulose-enzyme-microbe complex than when acting in the absence of cells.