R-(-)-linalool is widely used in pharmaceutical, agrochemical, and fragrance industries. However, plant extraction furnishes only limited and unstable R-(-)-linalool yields that do not satisfy market demand. Therefore, a sustainable yet efficient and productive method is urgently needed. To induce the R-(-)-linalool biosynthesis pathway in Escherichia coli, we expressed several heterologous (3R)-linalool synthases (LISs) and then chose a suitable LIS from Streptomyces clavuligerus (bLIS) for further study. The bLIS expression was markedly elevated by using optimized ribosomal binding sites and protein fusion tags. To increase the geranyl diphosphate content, we tested various alterations in prenyltransferases and their mutants. The final strain accumulated 100.1 and 1027.3 mg L-1 R-(-)-linalool under shake flask and fed-batch fermentation conditions, respectively. The latter is the highest reported R-(-)-linalool yield to date. This work could lay theoretical and empirical foundations for engineering terpenoid pathways and optimizing other metabolic pathways.