Genetic Manipulation with Genes for Xylose Isomerization

Historically, the earliest attempts to engineer xylose metabolic capabilities into S. cerevisiae involved the single gene for xylose isomerase (XI, catalyzing the intercon­version of xylose and xylulose) from bacteria (E. coli and B. subtilis), but these failed because the heterologous proteins produced in the yeast cells were enzymically inac- tive.4,69 A greater degree of success was achieved using the XI gene (xylA) from the bacterium Thermus thermophilus; the transformants could exhibit ethanol formation in O2-limited xylose fermentations.95

A crucial breakthrough, however, was made in 2003, when the gene encoding XI in the fungus Piromyces sp. strain E2 (capable of anaerobic growth on xylose) was recognized as part of the known “bacterial” pathway for xylose catabolism and, for the first time, was revealed to be functional in a eukaryote.96 The same research group at the Delft Technical University, The Netherlands, soon demonstrated that xylA gene expressed in S. cerevisiae gave high XI activity but could not by itself induce ethanol production with xylose as the carbon source.97 The additional genetic manipulations required for the construction of ethanologenic strains on xylose were the overexpression of XK, transketolase, transaldolase, ribulose 5-phosphate epim — erase, and ribulose 5-phosphate isomerase and the deletion of nonspecific AR, fol­lowed by selection of “spontaneous” mutants in xylose-limited continuous cultures and anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose media.98-100 The outcome was a strain with negligible accumulation of xylitol (or xylulose) and a specific ethanol production three — to fivefold higher than previously publicized strains.4 Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic cultures of the engineered strain in anaerobic batch culture, with glucose still being preferred as the carbon source.99

A side-by-side comparison of XR/XDH — and XI-based xylose utilizations in two isogenic strains of S. cerevisiae with genetic modifications to improve xylose metab­olism (overexpressed XK and nonoxidative pentose phosphate pathway enzymes and deleted AR) arrived, however, at widely different conclusions for the separately opti­mal parameters of ethanol production:101

• In chemically defined medium, the Xl-containing variant showed the high­est ethanol yield (i. e., conversion efficiency) from xylose.

• The XR/XDH transformant had the higher rate of xylose consumption, spe­cific ethanol production, and final ethanol concentration, despite accumu­lating xylitol.

• In a lignocellulose hydrolysate, neither transformant accumulated xylitol, but both were severely affected by toxic impurities in the industrially rel­evant medium, producing little or no ethanol, xylitol, or glycerol and con­suming little or no xylose, glucose, or mannose.

The bacterial XI gene from T. thermophilus was revisited in 2005 when this path­way for xylose utilization was expressed in S. cerevisiae along with overexpressed XK and nonoxidative pentose phosphate pathway genes and deleted AR; the engineered strain, despite its low measured XI activity, exhibited for the first time aerobic growth on xylose as sole carbon source and anaerobic ethanol production at 30°C.102