Xylitol Production by Quiescent E. Coli Cultures

1
Xylitol Production by Quiescent E. Coli Cultures
Geoffrey M. Geise - The Pennsylvania State
University
Department of Chemical Engineering Advisor Dr.
Patrick C. Cirino
Summary and Background Information
Project Goals
Results

• The Science Behind the Madness
• In biological systems, reduced cofactors such as
  NADH or NADPH can be used to effectively carry
  out oxidation/reduction reactions.
• During our experiments, cells are maintained in
  a Glucose-Xylose mixture. E. coli cells prefer
  to utilize glucose over other sugars. So,
  genetic modifications need to be made in order to
  allow xylose to enter the cells (to be reduced to
  xylitol) at the same time as glucose (used to
  regenerate reduced cofactors). Starting with the
  host organism E. coli W3110, metabolic
  engineering practices have allowed for the
  creation of additional strains. These strains
  can be created such that glucose and xylose can
  both be brought into by the cell at the same
  time. However, to ensure that the cells do not
  attempt to utilize xylose for other metabolic
  purposes, the xylB gene can be knocked out of the
  strains. This knockout prevents xylose from
  entering the pentose-phosphate pathway.
• Xylitol Production

The goal of this project is to study the
quiescent E. coli cell production of xylitol from
xylose. Different metabolic changes will be
imposed on the E. coli cells in an attempt to
maximize xylitol production. These changes will
include the use of different xylitol-producing
enzymes. In addition, attempts will be made to
increase the availability of reduced cofactors
within the cells.
Xylitol Production vs. Time for PC093815
Batch
Culture and Quiescent Cell Comparison
Experimental Procedure
Xylitol Production vs. Time - Strain/Plasmid
Comparison
Baffled Flask Cultures, 30mL Nitrogen Limited
Minimal Media (50mM Glucose, 300mM Xylose)

• Cultures maintained at 30?C
• Genes induced with 0.1?M IPTG
• Plasmids were maintained with Kanamycin
• As a control to ensure that protein synthesis has
  been disabled, chloramphenicol (Ch) was added

Xylitol Production vs. Time - sthA Gene Comparison
Baffled Flask Cultures, 30mL Nitrogen Limited
Minimal Media (50mM Glucose, 300mM Xylose)
Yield (Xylitol per Glucose Consumed) vs. Time -
Strain/Plasmid Comparison
Baffled Flask Cultures, 30mL Nitrogen Limited
Minimal Media (50mM Glucose, 300mM Xylose)

• Cultures were maintained at 30?C
• Genes were induced with 0.1?M IPTG
• Plasmids were maintained with Kanamycin
• As a control to ensure that protein synthesis has
  been disabled, chloramphenicol (Ch) was added

Strains and Plasmids Analyzed
Conclusions Comparison of quiescent cell data to
batch culture data indicated that the quiescent
cell method provides a more efficient way of
producing xylitol and a more systematic approach
to studying cofactor utilization. Comparison
of PC093815 and W31103815 data indicate that
the combination of the crp and ?xylB do indeed
increase the availability of NADPH for use in
xylitol production. More experiments will be run
to explore why the observed xylitol production in
PC073815 was lower than the wild-type
W3110-3815. Evidence from the PC09pPCC07 and
PC09pPCC17 experiment suggests that SthA may
increase xylitol production by increasing NADPH
levels within the cell. Future plans include
tests of a cbXR and sthA combination (plasmid
pPCC500). Yields are still lower than the
theoretical maximum, so there is still room for
improvement and future plans include attempts at
knocking out ATP pathways in an attempt to
increase xylitol production.