184
Chapter 5
parallel reaction to reaction 9 where the CoA is transferred to acetate rather than to butyrate.
Expand the stoichiometric calculation in b with these two reactions. Obviously, two more rates
have to be measured - but can you solve for the remaining rates? Discuss the result, referring to
the paper by Reardon
et al.
to see what they have done. Recalculate the result of b with reaction
15 instead of reaction 9.
Final note:
The present problem is very suitable for studies of pathway analysis in real industrial processes.
Both Papoutsakis (1984) and Reardon
et al.
(1987) have many suggestions for further variations of the
problem - e.g. abandoning the assumption of rNADH
= 0 or of
r
ATp = 0. The papers should be consulted for
these additional possibilities. In recent years a number of studies, especially in the group of Terry
Papoutsakis has shown that both yield (of solvents) and productivity can be improved by metabolic
engineering of the pathway. Hereby it may become economically viable to resurrect the old (lst World War)
process of making solvents by fermentation of house hold waste.
Problem
5.5 Production of propane 1,3-diol (3G) by fermentation
Zeng and Biebl (2002) have recently reviewed the possibilities of making 3G by a fermentation route.
The diol can be produced by several chemical routes from either ethylene (Shell) or from acrolein
(Degussa), but it may be cheaper to produce it by fermentation using either glycerol or the much cheaper
glucose as substrate. An enormous increase in demand for 3G
is envisaged, especially since it can
condense with terephtalic acid to form an excellent polyester (Sorona™ from DuPont).
3 G is a natural metabolic product in anaerobic cultivation of
Klebsiella pneumonica
on glycerol:
Glycerol —*■
3 Hydroxy propanal —>
1,3 Propane diol
(1)
The first step (a dehydration) is catalysed by glycerol dehydratase that requires the vitamin B
12
as co-
factor. Step 2 is catalysed by 1,3 propane diol dehydrogenase, and NADH is used as cofactor
When
Klebsiella
grows on glycerol the two steps (Fig. 2.4) from DHAP to glycerol are reversed. One
ATP and one NAD* is used per glycerol molecule. DHAP enters the EMP pathway and
via
pyruvate it is
metabolised to all the end products shown on Fig. 2.6A. (actually a pathway from pyruvate to 2,3 butane
diol
via
a-Acetolactate and acetoin can also be followed, but this will not be considered here.)
a.
Determine the maximum theoretical yield of 3G on glycerol by anaerobic fermentation with
Klebsiella.
The analysis should be based on the constraints rNADH
=
rAl? -
0. Which end
products of the mixed acids from pyruvate metabolism are desirable and which should if
possible be avoided?
b.
Klebsiella
is not the optimal production organism. In a series of brilliant metabolic
engineering studies (covered by many patents) DuPont and Genencor have succeeded to
insert the pathway from DHAP to glycerol (from
S. cerevisiae
) and from glycerol to 3G
(from
Klebsiella)
into
E.coli.
Hereby a well researched organism is used as host for
production of 3G and it has been shown that a very high titer of 129 g L
'1
3G can be
obtained by fed-batch fermentation of the engineered
E.coli
(Emptage
et al.,
2001).
We shall study the DuPont/Genencor process in the following. Three pathways are
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