Central Research Laboratory, Shenzhen Hospital of Integrated Traditional Chinese and Western Medicine, Shenzhen, China.
Dongguan Key Laboratory of Traditional Chinese Medicine and New Pharmaceutical Development, School of Pharmacy, Guangdong Medical University, Dongguan, China.
Appl Environ Microbiol. 2024 Jun 18;90(6):e0042924. doi: 10.1128/aem.00429-24. Epub 2024 May 23.
Microbial synthesis is a desirable approach to produce indirubin but suffers from low synthetic efficiency. Insufficient supply of reduced flavins is one major factor limiting synthetic efficiency. To address this, a novel flavin reductase, MoxB, was discovered through screening of the metagenomic library. MoxB showed a strong preference for NADH over NADPH as the electron source for FMN/FAD reduction and exhibited the highest activity at pH 8.0 and 30°C. It displayed remarkable thermostability by maintaining 80% of full activity after incubation at 60°C for 1 h. Furthermore, MoxB showed great organic solvent tolerance and its activity could be significantly increased by bivalent metal ions. In addition, heterologous expression of the gene in the indirubin-producing significantly improved indirubin production up to 15.12-fold. This discovery expands the understanding of flavin reductases and provides a promising catalytic tool for microbial indirubin production.IMPORTANCEMuch effort has been exerted to produce indirubin using engineered , but high-level production has not been achieved so far. Insufficient supply of reduced flavins is one key factor limiting the catalytic efficiency. However, the flavin reductases involved in indirubin biosynthesis have not been hitherto reported. Discovery of the novel flavin reductase MoxB provides a useful tool for enhancing indirubin production by . Overexpression of MoxB in indirubin-producing increased indirubin production by 15.12-fold in comparison to the control strain. Our results document the function of flavin reductase that reduces flavins during indirubin biosynthesis and provide an important foundation for using the flavin reductases to improve indirubin production by engineered microorganisms.
微生物合成是生产靛玉红的理想方法，但合成效率低。还原黄素的供应不足是限制合成效率的一个主要因素。为了解决这个问题，通过对宏基因组文库进行筛选，发现了一种新型黄素还原酶 MoxB。MoxB 对 NADH 的偏好强于 NADPH，作为 FMN/FAD 还原的电子供体，并在 pH8.0 和 30°C 时表现出最高的活性。它表现出显著的热稳定性，在 60°C 孵育 1 小时后保持 80%的全部活性。此外，MoxB 表现出很强的有机溶剂耐受性，其活性可以通过二价金属离子显著提高。此外，在产生靛玉红的 中异源表达 基因，可使靛玉红产量提高 15.12 倍。这一发现扩展了对黄素还原酶的理解，并为微生物靛玉红生产提供了有前途的催化工具。
尽管已经付出了很大的努力来使用工程 来生产靛玉红，但迄今为止还没有达到高水平的生产。还原黄素的供应不足是限制催化效率的一个关键因素。然而，参与靛玉红生物合成的黄素还原酶尚未被报道。新型黄素还原酶 MoxB 的发现为通过 增强靛玉红生产提供了有用的工具。与对照菌株相比，在产生靛玉红的 中过表达 MoxB 可使靛玉红产量提高 15.12 倍。我们的研究结果记录了在靛玉红生物合成过程中还原黄素的黄素还原酶的功能，并为利用黄素还原酶通过工程微生物提高靛玉红产量提供了重要基础。
