The bacterium K. oxytoca was isolated from paper and pulp mills and grows around other sources of wood; in addition to growing on hexoses and pentoses, it can uti­lize cellobiose and cellotriose but does not secrete endoglucanase.162 A University of Florida research group transformed strain M5A1 with the xylose-directing PET operon; unlike experience with E. coli, lower plasmid copy numbers gave higher ethanol productivity than with higher plasmid copy number.218 A PET transformant could produce ethanol at up to 98% of the theoretical yield and was highly suitable for lignocellulose substrates because it utilized xylose twice as fast as glucose — and twice as fast as did E. coli strain KO11.

Stabilizing the PET operon was accomplished by chromosomal integration at the site of the PFL (pfl gene); screening for mutants hyperresistant to the selectable chloramphenicol marker resulted in the P2 strain with improved fermentation kinetics capable of producing 44-45 g/l of ethanol from glucose or cellobiose (100 g/l) within 48 hr.219 Strain P2 has been demonstrated to generate ethanol from the cellulosic and lignocellulosic materials sugarcane bagasse, corn fiber, and sugarbeet pulp.167,220,221

As a candidate industrial strain for bioethanol production, P2 can utilize a wide range of low-molecular-weight substrates, including the disaccharides sucrose, malt­ose, cellobiose, and xylobiose, the trisaccharides raffinose, cellotriose, and xylotriose, and the tetrasaccharide stachyose.172,181,219,222 This relatively nonspecific diet has led to the cloning and expression of a two-gene K. oxytoca operon for xylodextrin utilization in E. coli strain KO11; the gene product of xynB is a xylosidase (which also has weak arabinosidase activity), whereas that of the adjacent gene in the K. oxytoca genome (xynT) is a membrane protein previously found in Na+/melibiose symporters[28] and related proteins functioning in transmembrane export and import.223 The enhanced recombinant E. coli could metabolize xylodextrins containing up to six xylose residues; unexpectedly, xylodextrin utilization was more rapid than by the donor K. oxytoca.