I. G. Fotheringham et al. / Bioorg. Med. Chem. 7 (1999) 2209±2213
2213
The PCR product was then cleaved with the enzymes
BamHI and SalI and the resulting 1.9 kB fragment was
ligated to the 4.76 kB fragment of pPOT3 (Nigel Grin-
ter) which was isolated by agarose gel electrophoresis
following similar cleavage by BamHI and SalI. The
resulting plasmid was named pPOT300.
Following incubation for 24 h a 200 mL sample was
taken and the cells removed by centrifugation. The
sample was then diluted 100-fold and subjected to
amino acid analysis by HPLC.
L-aspartic acid catabolism. One hundred miligrams wet
cell weight of W3110 and 100 mg wet cell weight of
HW1452 were added to a solution containing 800 mM
l-aspartic acid, adjusted to pH 7.5 with NaOH in a
2 mL reaction volume. The reaction was sealed and
shaken at 37ꢀC for 20 h.
Biosynthesis of L-2-aminobutyric acid
The eect of acetolactate synthase upon the eciency of
2-aminobutyrate biosynthesis was investigated using l-
aspartic acid and 2-ketobutyrate or l-aspartic acid and
l-threonine as substrates.
Following incubation for 24 h a 200 mL sample was
taken and the cells removed by centrifugation. The
sample was then diluted 100-fold and subjected to
amino acid analysis by HPLC.
Biosynthesis of L-2-aminobutyric acid using 2-keto-
butyrate and L-aspartic acid as substrates
HPLC analysis of amino acids
Reaction A. Two hundred milligrams wet cell weight of
W3110/pME64 was added to a solution containing
100 mM Tris±HCl pH 7.5, 500 mM 2-ketobutyrate and
500 mM l-aspartic acid, adjusted to pH 7.5 with NaOH
in a 2 mL reaction volume. The reaction was sealed and
shaken at 37ꢀC for 24 h. Following incubation for 24 h a
200 mL sample was taken and the cells removed by cen-
trifugation. The sample was then diluted 100 fold and
subjected to amino acid analysis by HPLC.
The amino acids were quanti®ed by HPLC using OPA/
BOC-Cys derivatization. The mobile phase used a gra-
dient of Pump A=60% MeOH, 40% 0.05M TEAP
buer pH=7. Pump B=H2O. Times: Pump B, 32% at
0 min, and at 6 min. By 8 min, 5% pump B. At 14.1 min
revert to starting conditions. Column heater @ 40ꢀC.
The column was a Supelcosil LC-18DB, 3 m, 150 Â
4.6 mm. The ¯ow rate was 1.0 mL/min, Detection UV
@ 338 nm, injection volume 10 L.
Reaction B. Two hundred milligrams wet cell weight of
W3110/pME64 and 100 mg wet cell weight of W3110/
pPOT300 were added to a solution containing 100 mM
Tris±HCl pH 7.5, 500 mM 2-ketobutyrate and 500 mM
l-aspartic acid, adjusted to pH 7.5 with NaOH in a
2 mL reaction volume. The reaction was sealed and
shaken at 37ꢀC for 24 h.
Acknowledgements
We would like to thank Monsanto Corporation for
provision of excellent research facilities for this work.
We also wish to thank L. Bartelt and G. Singh for pro-
viding the analytical support for this work.
Following incubation for 24 h a 200 mL sample was
taken and the cells removed by centrifugation. The
sample was then diluted 100 fold and subjected to
amino acid analysis by HPLC.
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Biosynthesis of L-2-aminobutyrate using L-threonine and
L-aspartate as substrates.
Reaction C. Two hundred milligrams wet cell weight of
W3110/pME64 and 100 mg wet cell weight of W3110/
pIF347 were added to a solution containing 500 mM l-
threonine and 500 mM L-aspartic acid, adjusted to pH 8
with NaOH in a 2 mL reaction volume. The reaction
was sealed and shaken at 37ꢀC for 24 h.
Following incubation for 24 h a 200 mL sample was
taken and the cells removed by centrifugation. The
sample was then diluted 100-fold and subjected to
amino acid analysis by HPLC.
Reaction D. Two hundred milligrams wet cell weight of
W3110/pME64, 100 mg wet cell weight of W3110/
pIF347 and 100 mg/mL W3110/pPOT300 were added to
a solution containing 500 mM l-threonine and 500 mM
l-aspartic acid, adjusted to pH 8 with NaOH in a 2 mL
reaction volume. The reaction was sealed and shaken at
37ꢀC for 24 h.
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