Site-specific recombinases are a family of DNA-modifying enzymes that can recognize short specific DNA sequences and drive recombination between them to rearrange DNA fragments which results in excision, integration or inversion. Here I describe in vivo application of the recombinases for chassis optimization for heterologous pathway expression in synthetic yeast and in vitro application of recombinases for gene expression optimization. For in vivo application, two recombination systems, Cre/loxP and Dre/rox, were developed for driving orthogonal co-SCRaMbLE of normal synthetic chromosomes and the tRNA Neochromosome. The functions of two recombinases were engineered to be activated and controlled by two hormone molecules, b-estradiol and RU486. In addition, a SCRaMbLE-in device was designed to integrate a pathway of interest into a synthetic chromosome in yeast while driving the normal SCRaMbLE process at the same time for chassis level optimization by the Cre/loxP recombination system. For in vitro application, three recombinases, Cre, Dre and VCre, were explored to integrate promoters into a pathway of interest for gene expression level diversification in metabolic engineering. To attempt broader application of the recombinases for metabolic engineering, a side project in metabolic engineering was also involved in my PhD study. As part of a collaborative Synthetic Natural Product (SynNP) project applying synthetic biology to discover and design new antibiotics against tuberculosis and other infectious diseases, a YeastFab compatible assembly method was designed for large pathway construction and tested for heterologous expression of the RiPPs exemplar pathway of nocathiacin I in S. cerevisiae.