National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China.
J Biotechnol. 2020 Sep 10;321:68-77. doi: 10.1016/j.jbiotec.2020.05.009. Epub 2020 May 20.
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.
磷酸吡哆醛（PLP）是一种必需的辅因子，参与了约 4%的酶促反应，这些反应被酶委员会分类。细胞内的 PLP 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 辅因子的自给自足，构建了从头核糖 5-磷酸（R5P）依赖性 PLP 合成途径。将枯草芽孢杆菌 168 中的 pdxST 基因引入酪氨酸苯酚解酶（TPL）过表达的大肠杆菌 BL21（DE3）菌株中。TPL 和 PdxST 与双启动子或兼容的双质粒系统一起共表达。当这些菌株中 TPL 和 PdxST 共表达时，3,4-二羟基苯乙酸-L-丙氨酸（L-DOPA）的产量并没有随着细胞内 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 产量，并避免 L-DOPA-PLP 环状加合物的形成。硫苷开关与 PLP 结合形成复合物，使核糖体无法接触到 Shine-Dalgarno（SD）序列。因此，这种代谢物感应调节系统被应用于调节 pdxST mRNA 的翻译。将 riboswitch 引入 pET-TPL-pdxST-2 中，通过隔离核糖体结合位点，在翻译水平下调 PdxST 的表达和 PLP 的生物合成。结果，使用菌株 BL21-TPLST-Ribo1 的 L-DOPA 的产量和生产率分别提高到 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 24.8 μM。
