产S-雌马酚梭菌C1三代全基因组测序及功能基因筛选鉴定

Whole genome sequencing and functional gene screening of S-equol-producing Clostridium sp. C1

摘要:

【目的】挖掘产S-雌马酚梭菌C1转化大豆苷元产生S-雌马酚的功能基因，为梭菌C1的S-雌马酚转化机制研究提供参考，并为利用合成生物学方法生产S-雌马酚提供新基因资源。【方法】利用GridION测序平台，对梭菌C1进行第三代全基因组测序、基因组组装和功能注释等分析，从C1菌全基因组中筛选和鉴定参与S-雌马酚生物转化的功能基因。【结果】C1全基因组大小为3 035 113 bp，预测编码3 166个基因，包含53个tRNA、15个rRNA、4个ncRNA和1个基因岛。通过生物信息学分析，发现C1-07020基因编码蛋白与已报道的Lactococcus sp.20-92大豆苷元还原酶具有44.8%的氨基酸序列相似性和相同的3个功能保守结构域，体外蛋白功能验证表明，C1-07020具有大豆苷元还原酶功能。此外，C1菌中没有发现与已知产S-雌马酚菌相似的功能基因簇或大豆苷元还原酶以外的其他功能基因。【结论】在C1中鉴定到一个新的产S-雌马酚功能基因，并发现了C1可能具备特殊的产S-雌马酚机制，实验所获基础数据可为进一步挖掘产S-雌马酚新功能基因、了解S-雌马酚的生成机制及体外产S-雌马酚基因资源的应用提供参考。

Abstract:

[Objective] This paper aims to investigate genes related to the production of S-equol from daidzein in Clostridium sp. C1, which is expected to serve as a reference for exploring the S-equol transformation mechanism in C1 and discover genes for the synthesis of S-equol by synthetic biology techniques.[Methods] Through the third-generation sequencing (GridION), assembly, and function annotation of the whole genome of C1, genes related to the biotransformation of S-equol were screened and identified.[Results] The genome was 3 035 113 bp, with 3 166 protein-coding genes, 53 tRNA genes, 15 rRNA genes, 4 ncRNA genes, and 1 genomic island. Bioinformatics analysis revealed that C1-07020 protein shared 44.8% amino acids and 3 conserved domains with daidzein reductase of Lactococcus sp. 20-92. In vitro verification showed that C1-07020 protein had similar functions to daidzein reductase. In addition, no other S-equol-producing gene clusters or other functional genes were identified from C1, which suggests that C1 may have a different metabolic mechanism from other S-equol-producing bacteria.[Conclusion] One S-equol-producing gene was identified from C1 and C1 might have a unique S-equol synthesis mechanism. The results of this study can serve as a reference for further exploring S-equol-producing genes, the production mechanism, and in vitro utilization of the genes.