Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures

Abstract

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.

FIG. 1

Schematic flow sheet for the construction of the integrating vector YIpXR/XDH/XK harboring XYL1, XYL2, and XKS1. XYL1 is under control of the ADH promoter and terminator, whereas both XYL2 and XKS1 are under control of the PGK promoter and terminator. (a) Partial digestion of pXks with XmnI and PvuII to remove TRP1 and the 2μ origin of replication, followed by recircularization of the remaining fragment to create YpXK. (b) Partial digestion of YpXK with HindIII and BamHI. (c) Excision of XYL1 and XYL2 from pY7 by partial digestion with HindIII and BamHI. (d) Excision of the HIS3 cassette from YDp-H with BamHI. (e) Ligation of the three fragments to create YIpXR/XDH/XK. This integrating vector was linearized with PstI to target integration to HIS3 in the chromosome. The restriction sites are labeled as follows: B, BamHI; H, HindIII; Ps, PstI; Pv, PvuII; X, XmnI; and X/Pv, hybrid site of XmnI and PvuII. Only relevant restriction sites are shown for each step, and those that were used are in bold.

FIG. 2

Xylose uptake rate at different xylose concentrations in the feed during anaerobic fermentation of xylose-glucose mixtures with S. cerevisiae TMB 3001. The equation for the line is y = 2.266 + 0.349x, and the standard error of the estimate is 0.5298.