Li Hailiang, Mao Shaoming, Chen Huahai, Zhu Liying, Liu Wei, Wang Xin, Yin Yeshi
Key Laboratory of Comprehensive Utilization of Advantage Plants Resources in Hunan South, College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou, China.
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
Front Microbiol. 2018 Jun 4;9:1182. doi: 10.3389/fmicb.2018.01182. eCollection 2018.
()-equol is one of the major metabolites of daidzein that is produced by human and animal gut bacteria. Most of the physiological functions of soybean isoflavones, such as anti-oxidative activity, anti-cancer activity, and cardiovascular protection have been ascribed to ()-equol. However, only 30-50% people contain this kind of equol-producing bacteria, and therefore are able to convert daidzein to ()-equol. Administration of ()-equol may be more beneficial than soybean isoflavones. The aim of this study was to construct an engineered ()-equol resistant to enhance ()-equol production . First, transposon mutagenesis libraries were constructed and screened to isolate the ()-equol resistant mutant strain BL21 () in order to overcome the inhibitory effects of ()-equol on bacterial growth. Bacterial full genome scan sequencing and overexpression results revealed that the gene was responsible for this resistance. Second, the ()-equol-producing genes L-, L-, L-, and L- of strain 20-92 were synthesized and cloned into compatible vectors, pETDuet-1 and pCDFDuet-1. These plasmids were subsequently transformed into BL21 (DE3) and its mutant BL21 (). Both engineered BL21 (DE3) and BL21 () could use daidzein as substrate to produce ()-equol under both anaerobic and aerobic conditions. As expected, engineered BL21 () had faster growth rates than BL21 (DE3) when supplemented with high concentrations of ()-equol. The yield and the daidzein utilization ratio were higher for engineered BL21 (). Interestingly, engineered BL21 () was able to convert daidzein to ()-equol efficiently under aerobic conditions, providing a convenient method for ()-equol production . In addition, a two-step method was developed to produce ()-equol using daidzin as substrate.
（）-雌马酚是大豆苷元的主要代谢产物之一，由人和动物肠道细菌产生。大豆异黄酮的大多数生理功能，如抗氧化活性、抗癌活性和心血管保护作用，都归因于（）-雌马酚。然而，只有30%-50%的人含有这种能产生雌马酚的细菌，因此能够将大豆苷元转化为（）-雌马酚。服用（）-雌马酚可能比大豆异黄酮更有益。本研究的目的是构建一种工程化的（）-雌马酚抗性菌株以提高（）-雌马酚的产量。首先，构建转座子诱变文库并进行筛选，以分离出（）-雌马酚抗性突变菌株BL21（），从而克服（）-雌马酚对细菌生长的抑制作用。细菌全基因组扫描测序和过表达结果表明该基因负责这种抗性。其次，合成了菌株20-92的（）-雌马酚产生基因L-、L-、L-和L-，并将其克隆到兼容载体pETDuet-1和pCDFDuet-1中。随后将这些质粒转化到BL21（DE3）及其突变体BL21（）中。工程化的BL21（DE3）和BL21（）在厌氧和好氧条件下都能以大豆苷元为底物产生（）-雌马酚。正如预期的那样，当添加高浓度的（）-雌马酚时，工程化的BL21（）的生长速度比BL21（DE3）更快。工程化的BL21（）的产量和大豆苷元利用率更高。有趣的是，工程化的BL21（）能够在有氧条件下有效地将大豆苷元转化为（）-雌马酚，为（）-雌马酚的生产提供了一种便捷的方法。此外，还开发了一种以黄豆苷为底物生产（）-雌马酚的两步法。
