Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.
After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.
Kuyper
Marko
M
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Hartog
Miranda M P
MM
Toirkens
Maurice J
MJ
Almering
Marinka J H
MJ
Winkler
Aaron A
AA
van Dijken
Johannes P
JP
Pronk
Jack T
JT
D019747:Aldose-Ketose Isomerases / Q000235:genetics* / Q000378:metabolism*; D000693:Anaerobiosis; D003470:Culture Media; D005285:Fermentation; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D007218:Industrial Microbiology; D020411:Oligonucleotide Array Sequence Analysis; D010427:Pentose Phosphate Pathway; D020089:Piromyces / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014994:Xylose / Q000378:metabolism*
D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose