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 Shine-Dalgarno (SD) sequence. Therefore, this metabolite-sensing regulation system was applied to regulate the translation of pdxST 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.

吡rid醛5'-磷酸（PLP）是必不可少的辅助因子，参与酶委员会规定的约4％的酶促活性。PLP的细胞内水平通常低于工业催化所需的水平。为了实现全细胞生物转化中PLP辅因子的自给，构建了从头核糖5磷酸（R5P）依赖的PLP合成途径。来自枯草芽孢杆菌168的pdxST基因被引入到酪氨酸酚裂解酶（TPL）过表达的大肠杆菌中BL21（DE3）菌株。TPL和PdxST与双启动子或兼容的双质粒系统共表达。在具有TPL和PdxST共表达的这些菌株中，3,4-二羟基苯乙酸酯-L-丙氨酸（L-DOPA）滴度没有随细胞内PLP浓度的增加而增加。因此，有必要优化细胞内PLP代谢水平，以获得更高的L-DOPA滴度，并避免形成L-DOPA-PLP环状加合物。所述THI核糖开关结合于PLP和形式的复合物，使得核糖体不能访问的Shine-Dalgarno（SD）序列。因此，该代谢物传感调控系统被用于调控pdxST的翻译mRNA。Riboswitch被引入pET–TPL–pdxST-2中，以通过隔离核糖体结合位点在翻译水平上下调PdxST的表达和PLP的生物合成。结果，使用BL21–TPLST–Ribo1菌株的L-DOPA的效价和生产率分别提高至69.8 g / L和13.96 g / L / h，儿茶酚转化率为95.9％，细胞内PLP积累为24.8μM 。