Trehalose is a nonreducing disaccharide and has a wide range of applications in food and biorelated industry. This sugar can be synthesized from maltose in one step by trehalose synthase. In this study, we attempted to overproduce trehalose synthase from Picrophilus torridus (PTTS), a thermoacidophilic archaea, in Escherichia coli . However, overproduction of PTTS was hampered when the T7 promoter-driven PTTS gene (PT7-PTTS) on a multicopy plasmid was employed in E. coli . The factors limiting PTTS production were identified in a systematic way, including the codon bias, plasmid instability, a redundant gene copy, a high basal level of PTTS, and metabolic burden resulting from the mutlicopy plasmid DNA and antibiotics. To overcome these difficulties, an E. coli strain was developed with insertion of PT7-PTTS into the chromosome and enhanced expression of genomic argU tRNA and ileX tRNA genes. Without the selective pressure, the constructed producer strain was able to produce a stable and high-level production of recombinant PTTS. Overall, we proposed a simple and effective method to address the issue that is most commonly raised in overproduction of heterologous proteins by E. coli .