Fermentation of d-Xylose to Ethanol by Genetically Modified Klebsiella planticola

d-Xylose is a plentiful pentose sugar derived from agricultural or forest residues. Enteric bacteria such as Klebsiella spp. ferment d-xylose to form mixed acids and butanediol in addition to ethanol. Thus the ethanol yield is normally low. Zymomonas spp. and most yeasts are unable to ferment xylose, but they do ferment hexose sugars to ethanol in high yield because they contain pyruvate decarboxylase (EC 4.1.1.1), a key enzyme that is absent from enteric bacteria. This report describes the fermentation of d-xylose by Klebsiella planticola ATCC 33531 bearing multicopy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene markedly increased the yield of ethanol to 1.3 mol/mol of xylose, or 25.1 g/liter. Concurrently, there were significant decreases in the yields of formate, acetate, lactate, and butanediol. Transconjugant Klebsiella spp. grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even in the absence of selective pressure by antibiotics to maintain the plasmid. Ethanol production was 31.6 g/liter from 79.6 g of mixed substrate per liter chosen to simulate hydrolyzed hemicellulose. The physiology of the wild-type of K. planticola is described in more detail than in the original report of its isolation.