Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China.
Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China.
Int J Mol Sci. 2023 Oct 16;24(20):15214. doi: 10.3390/ijms242015214.
Tetrahydroprotoberberines (THPBs) are plant-specific alkaloids with significant medicinal value. They are present in trace amounts in plants and are difficult to chemically synthesize due to stereoselectivity and an unfavorable environment. In this study, a selective methylation strategy was developed for the biocatalysis of seven high-value-added THPB compounds using 4'--methyltransferase (), norcoclaurine 6--methyltransferase (), and ()-scoulerine 9--methyltransferase ( and ) in engineered . The methyltransferases , , , and were expressed heterologously in . Compound (10-methoxy-2,3,9-tetrahydroxyberbine) was synthesized using the recombinant strain and the substrate 2,3,9,10-tetrahydroxyberbine. Compound (9-methoxy-2,3,10-tetrahydroxyberbine) was produced in the recombinant () strain , and compounds and (discretamine) were produced in the recombinant strain . Compounds (9,10-methoxy-2,3-tetrahydroxyberbine) and (corypalmine) were obtained by co-culturing the recombinant strains and with substrate. Compounds (scoulerine) and (isoscoulerine) were produced by co-culturing the substrate with the recombinant strains and . To increase the yield of novel compound , the flask culture conditions of the engineered strain were optimized, resulting in the production of 165.74 mg/L of this compound. This study thus presents an enzymatic approach to the synthesis of high-value-added THPBs with minimum environmental wastage.
四氢原小檗碱（THPBs）是具有重要药用价值的植物特异性生物碱。它们在植物中以痕量存在，由于立体选择性和不利的环境，难以通过化学合成得到。在这项研究中，使用 4′-甲基转移酶（）、诺卡枯林 6′-甲基转移酶（）和（）-胡椒碱 9′-甲基转移酶（和），针对七种高附加值 THPB 化合物，开发了一种选择性甲基化的生物催化策略，在工程菌中表达。甲基转移酶、、、和在中异源表达。使用重组菌株和底物 2,3,9,10-四羟基小檗碱合成化合物（10-甲氧基-2,3,9-四氢小檗碱）。化合物（9-甲氧基-2,3,10-四羟基小檗碱）在重组（）菌株中产生，化合物和（离散胺）在重组菌株中产生。通过共培养重组菌株和底物，获得化合物（9,10-甲氧基-2,3-四羟基小檗碱）和（科里巴明）。通过共培养底物和重组菌株，获得化合物（胡椒碱）和（异胡椒碱）。为了提高新型化合物的产量，优化了工程菌的摇瓶培养条件，最终该化合物的产量达到 165.74mg/L。因此，本研究提出了一种酶法合成高附加值 THPBs 的方法，最大限度地减少了环境浪费。
