Optimization of Xylose Fermentation

1
Optimization of Xylose Fermentation
NISHANT KUMAR M.Sc- ii Roll no 06530006
2
INTRODUCTION

• Xylose or wood sugar, one of the most abundant
  carbohydrates in nature, is an aldopentose a
  monosaccharide containing five carbon atoms and
  including an aldehyde functional group.

3
XYLOSE METABOLISM
4
Xylose Metabolism S. cerevisiae

• Native strains of S. cerevisiae do not assimilate
  xylose.
• In xylose fermenting yeasts, xylose is reduced to
  xylitol by NADPH-linked xylose reductase (XR) and
  xylitol is oxidized to xylulose by NAD-linked
  xylitol dehydrogenase (XDH). Subsequently
  xylulose is phosphorylated by xylulokinase (XK)
  for assimilation via the pentose phosphate
  pathway.

5
Xylose Metabolism S. cerevisiae

• The discovery that S. cerevisiae can ferment
  xylulose initiated metabolic engineering of
  xylose fermentation in S. cerevisiae.
• Heterologous expression of genes (XYL1, XYL2, and
  XYL3) coding for XR, XDH, and XK from P. stipitis
  enabled S. cerevisiae to utilize xylose as a sole
  carbon source.

6
Shuffling of Promoters for Multiple Genes To
Optimize XyloseFermentation in an Engineered
Saccharomyces cerevisiae Strain

• Chenfeng Lu and Thomas Jeffries
• APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct.
  2007, p. 60726077

7
Multiple-gene-promoter shuffling (MGPS)
8
Fuse selected promotor to target genes
9
Concatenate each promoter-gene product with
different combinations
10
Screen for phenotype of interest among these
transformant
11
AIM

• Optimization of xylose fermentation by shuffling
  the promoters for GND2 and HXK2 with the genes
  for transaldolase (TAL1), transketolase (TKL1),
  and pyruvate kinase (PYK1) in the Saccharomyces
  cerevisiae strain FPL-YSX3.

12
Selection of promoters

• The promoters for this study were chosen on the
  basis of the native gene expression that they
  control promotor strength was validated by
  using lacZ expression ß-Gal assay.

13
Correlation between expression array data and
ß-Gal activitiesresulting from promoter
strengths of selected genes.
14
(No Transcript)
15
RESULTS
16
RESULTS.
17
CONCLUSION

• The optimal expression levels for TAL1,TKL1, and
  PYK1 were identified by analysis of volumetric
  ethanol production by transformed cells the
  optimal combination for ethanol production was
  found to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1

18

• THANX!!!!