Page 357 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

15.3 Cascade Reactions for Assaying Transketolase Activity by In Vivo Selection 333

First, it was established that expression of the yeast TKL1 gene in E. coli led to a
functional TK. E. coli auxotrophs expressing the yeast TKL1 gene were then used
to convert the synthetic compounds 16a and 16b into a usable amino acid source.
(−)
For that purpose, leucine and methionine auxotrophic mutants (Leu (−) and Met )
devoid of the chromosomal TK-encoding genes were constructed and transformed
(+)
(−)
with a TKL1-harboring plasmid (strain TK ) or empty vector (strain TK ). As
shown in Tables 15.3 and 15.4, leucine and methionine equally met the metabolic
requirement of both TK-deficient and TK-expressing bacteria.
TK-expressing methionine auxotrophs displayed similar generation times when
either compound 16b or methionine were supplied in the culture medium. By
contrast, compound 16b was less efficient than methionine in TK-deficient bacteria
(Table 15.4). Thus the growth of the methionine auxotroph with compound 16b
seems directly correlated with the expression of yeast TK. However, the generation
time displayed by the TK-deficient methionine auxotroph indicates that compound
16b was still converted into methionine by a TK-independent pathway. The results
obtained with leucine auxotrophs indicate that compound 16a was a poor source of

Table 15.3 Growth of leucine E. coli auxotrophs.
Genotype Generation time for Leu auxotrophs cultivated with
indicated source of Leu
Leu (0.25 mM) 16a (2.5 mM)

Leu (−) TK (+) 6 h 30 min 125 h
Leu (−) TK (−) 4 h 40 min 700 h

+
(+)
Leu (−) TK : ΔtktA ΔtktB, ΔleuABCD, pDRI5 TKL1 .
(−)
Leu (−) TK : ΔtktA ΔtktB, ΔleuABCD, empty vector pSP100.
a Culture medium: MS glucose 0.2% supplemented with phenylalanine, tyrosine,
and tryptophan (0.3 mM each), shikimic acid (0.3 mM), pyridoxine (3 μM), and the
indicated amino acid source.
Table 15.4 Growth of methionine E. coli auxotrophs.

Genotype Generation time for met auxotrophs cultivated with
indicated source of met
Met (0.25 mM) 16b (2.5 mM)

Met (−) TK (+) 3 h 45 min 6 h
Met (−) TK (−) 5 h 45 min 20 h 15 min

(+)
(+)
Met (−) TK : ΔtktA ΔtktB, ΔmetA, pGEN377 TKL1 .
(−)
Met (−) TK : ΔtktA ΔtktB, ΔmetA, empty vector pVDM18.
a
Culture medium: MS glucose 0.2% supplemented with phenylalanine, tyrosine,
and tryptophan (0.3 mM each), shikimic acid (0.3 mM), pyridoxine (3 μM), and the
indicated amino acid source.