Ergothioneine, a histidine-derived thiol, protects cells against reactive oxygen species and is emerging as a longevity vitamin. Ovothiol, another histidine-derived thiol, is also a potent antioxidant with therapeutic potential due to its anti-inflammatory and anti-proliferative activities. Despite these promising health benefits, the production of ergothioneine is limited by the underlying challenges of its only industrial synthetic method, while ovothiol is not commercially available. Due to these issues, the production of these thiols through metabolic engineering/synthetic biology approaches is appealing. The central steps in the ergothioneine and ovothiol biosynthetic pathways are the oxidative coupling C-S bond formation reaction mediated by non-heme iron sulfoxide synthases, and the pyridoxal-5'-phosphate (PLP)-dependent C-S lyases. This sulfur transfer strategy differs from all other pathways reported. Therefore, these trans-sulfuration reactions in ergothioneine and ovothiol biosyntheses are significant from both basic and translational research perspectives, hence, they were selected as my thesis project. This thesis comprises of five chapters. Sulfur metabolism and the biosynthesis of sulfur-containing natural products are presented in Chapter 1. The computational-guided protein engineering of a thermophilic sulfoxide synthase (EgtB) from Chloracidobacterium thermophiluim is covered in Chapter 2. Chapter 3 describes the mechanistic studies of the reductive C-S lyase (Egt2 from the Neurospora crassa’s ergothioneine biosynthesis), which revealed the involvement of a sulfenic acid intermediate in this reaction. In addition to reconstituting the ergothioneine biosynthetic pathway in vitro presented in Chapter 3, I fully reconstituted the in vitro ovothiol A biosynthetic pathway from Erwinia tasmaniensis, which is described in Chapter 4. In Chapter 5, the mechanistic studies of the ovothiol sulfoxide synthase OvoA using unnatural amino acid incorporation via amber-codon suppression are discussed. The success of this thesis work paves the way for the industrial production of ergothioneine and ovothiol through metabolic engineering/synthetic biology approaches. This study has also laid the foundation for future in-depth mechanistic characterization of these novel enzymes.