Biomedical Engineering Reference
In-Depth Information
(a)
Acetobacter
suboxydans
Chemical
D
-glucose
D
-sorbitol
L
-sorbose
Diacetone
-2 keto-
L
-
gulonic acid
Chemical
Chemical
2 keto-
L
-
gulonic acid
Ascorbic acid
(b)
CHO
COOH
COOH
OH
O
O
2,5 DKG
reductase
Endogenous
HO
HO
HO
Erwinia
enzymes
(Corynebacterium)
OH
OH
OH
Fig. 14.17
Simplified route to vitamin
C (ascorbic acid) developed by cloning
in
Erwinia
the
Corynebacterium
gene for
2,5-diketogluconic acid reductase.
(a) Classical route to vitamin C. (b) The
simplified route to 2-ketogulonic acid,
the immediate precursor of vitamin C.
OH
O
HO
CH
2
OH
D
-glucose
CH
2
OH
2,5 diketo-
D
-
gluconic acid
CH
2
OH
2-keto-
L
-
gulonic acid
burgundy colour. To make textile-quality indigo,
there must be no indirubin present. Screening soil
microorganisms with the capacity to degrade indole
resulted in the identification of an enzyme, isatin
hydrolase, that can degrade isatin to isatic acid.
After cloning the gene for isatin hydrolase in the
indigo overproducing strains, the indigo product
obtained performed as well as chemically produced
material.
A slightly different approach to that above has
yielded a new route to vitamin C. The conventional
process starts with glucose and comprises one
mirobiological and four chemical steps (Fig. 14.17).
By cloning in
Erwinia
a single gene - that from
Corynebacterium
encoding 2,5-diketogluconic acid
reductase - the process can be simplified to a single
microbiological and a single chemical step (Ander-
son
et al.
1985). After observations of unexpectedly
low yields of 2-ketogulonic acid in the recombinant
strain, it was found that 2-ketogulonic acid was
converted to l-idonic acid by an endogenous 2-
ketoaldonate reductase. Cloning, deletion mutagen-
esis and homologous recombination of the mutated
reductase gene into the chromosome were some
of the several steps taken to develop an organism
capable of accumulating large amounts (120 g/l) of
2-ketogulonic acid (Lazarus
et al.
1990). So far,
attempts to manufacture vitamin C directly from
glucose have been unsuccessful. However, enzymes
that can convert 2-ketogulonic acid to ascorbic acid
have been identified and the objective now is to
clone these activities into
Erwinia
(Chotani
et al.
2000).
Combinatorial biosynthesis
A number of widely used antibiotics and immuno-
suppressants belong to a class of molecules known
