Economically viable ethanol production from the hemicellulose fraction of plant biomass requires the simultaneous conversion of both pentoses and hexoses at comparable rates and with high yields. Yeasts, in particular Saccharomyces spp., are the most appropriate candidates for this process since they can grow fast on hexoses, both aerobically and anaerobically. Furthermore, they are much more resistant to the toxic environment of lignocellulose hydrolysates than (genetically modified) bacteria.
Although wild-type S. cerevisiae strains can slowly metabolise the pentose sugar xylulose, they are not capable of metabolising xylose. Already in the 1980's it was suggested that metabolic engineering of yeasts for xylose utilization should be based on the introduction of xylose isomerase (XI, EC 5.3.1.5) rather than expressing heterologous xylose reductase and xylitol dehydrogenase to convert xylose into xylulose. Unfortunately, all attempts of introducing a bacterial xylose isomerase in S. cerevisiae have failed to produce a functionally expressed xylose isomerase with the notable exception of the T. thermophilus isomerase. This enzyme was functionally expressed in S. cerevisiae but only very low activities were observed at growth permitting temperatures. This situation drastically changed when a newly discovered xylose isomerase from the anaerobic fungus Piromyces Sp.E2 was introduced in S. cerevisiae and high levels of enzyme activities were observed enabling this strain to grow anaerobically and produce ethanol from xylose (WO 03/062430 and WO 06/009434). Such yeast strains for the first time provided specific rates of xylose consumption and ethanol formation that are compatible with ethanol production at a commercial scale.
Since the discovery of the functional expression of the Piromyces xylose isomerase in yeast several reports have appeared of functional expression in yeasts of other xylose isomerases, all of which share more than 70% amino acid sequence identity with the Piromyces enzyme, such e.g. the bacterial xylose isomerase from Bacteroides (WO 04/099381; WO 06/009434; WO 09/109633), and the fungal xylose isomerases from Cyllamyces (WO 04/099381) and Orpinomyces (Madhavan et al., 2008, DOI 10.1007/s00253-008-1794-6).
However, prior to Dec. 24, 2008 no reports have issued of functional expression in yeasts of xylose isomerases having less than 70% amino acid sequence identity with the Piromyces enzyme. More recently, in February 2009, Brat et al. (2009, Appl. Environ. Microbiol. 75: 2304-2311) published functional expression in the yeast S. cerevisiae of a xylose isomerise from the anaerobic bacterium Clostridium phytofermentans, the amino acid sequence of which shares only 52% identity with that of the Piromyces enzyme.
To date some 450 xylose isomerase amino acid sequences are publicly available in Genbank and other sequence databases, including the xylose isomerase sequences of Piromyces, Cyllamyces aberensis, Physcomitrella patens, Arabidopsis thaliana, Haemophilus somnus, Ciona intestinalis, Clostridium difficile, Thermatoga maritime, Bacteroides fragilis, Burkholderia phytofirmans, Arthrobacter aurescens and Fusobacterium mortiferum. 
There is, however, still a need in the art for nucleotide sequences encoding other xylose isomerases that may be used to transform host cells like S. cerevisiae to confer to them the ability of isomerising xylose to xylulose, so as to enable the use of thus transformed host cell in processes for the production of ethanol or other fermentation products by fermentation of pentose-containing feedstock.
USPTO Backgrounds
USPTO Backgrounds