Regulating the biosynthesis of pyridoxal 5’-phosphate with riboswitch to
enhance L-DOPA production by Escherichia coli whole-cell
biotransformation
Abstract
Pyridoxal 5’-phosphate (PLP) is an essential cofactor that participates
in ~4% enzymatic activities cataloged by the Enzyme
Commission. The intracellular level of PLP is usually lower than that
demanded in industrial catalysis. To realize the self-supply of PLP
cofactor in whole-cell biotransformation, the de novo ribose
5-phosphate (R5P)-dependent PLP synthesis pathway was constructed. The
pdxST genes from Bacillus subtilis 168 were introduced into the
tyrosine phenol-lyase (TPL)-overexpressing Escherichia coli
BL21(DE3) strain. TPL and PdxST were co-expressed with a double-promoter
or a compatible double-plasmid system. The
3,4-dihydroxyphenylacetate-L-alanine (L-DOPA) titer did not increase
with the increase in the intracellular PLP concentration in these
strains with TPL and PdxST co-expression. Therefore, it is necessary to
optimize the intracellular PLP metabolism level so as to achieve a
higher L-DOPA titer and avoid the formation of L-DOPA–PLP cyclic
adducts. The thi riboswitch binds to PLP and forms a complex such
that the ribosome cannot have access to the SD sequence. Therefore, this
metabolite-sensing regulation system was applied to reduce the
translation of mRNA. Riboswitch was introduced into pET–TPL–pdxST-2 to
downregulate the expression of PdxST and biosynthesis of PLP at the
translation level by sequestering the ribosome-binding site. As a
result, the titer and productivity of L-DOPA using the strain
BL21–TPLST–Ribo1 improved to 69.8 g/L and 13.96 g/L/h, respectively,
with a catechol conversion of 95.9% and intracellular PLP accumulation
of 24.8 µM.