Article

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering.

Department of Applied Microbiology, Lund University, PO Box 124, SE-22100 Lund, Sweden.
Yeast (impact factor: 1.89). 05/2005; 22(5):359-68. DOI:10.1002/yea.1216 pp.359-68
Source: PubMed

ABSTRACT A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.

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Keywords

30 degrees C
 
aerobic xylose growth
 
aerobic xylose growth rate
 
aldose reductase
 
efficient xylose utilization
 
genes encoding xylulokinase
 
GRE3 gene coding
 
metabolic steps downstream
 
multicopy plasmid
 
multiple genetic modifications
 
non-oxidative pentose phosphate pathway
 
PPP genes
 
ribose 5-phosphate ketol-isomerase
 
ribulose 5-phosphate epimerase
 
Thermus thermophilus
 
XDH genes chromosomally
 
XI)-expressing strains
 
xylose reductase
 
xylose utilization
 
xylose-growing
 

Kaisa Karhumaa