Efficient production of α-pinene through identifying the rate-limiting enzymes and tailoring inactive terminal of pinene synthase in Escherichia coli

α-Pinene has been indicated as an ideal biofuel or fuel precursors. The higher energy value and broad application of pinene dimers has recently been evaluated as a promising high-density jet fuel. In this work, the microbial de novo synthesis of α-pinene in Escherichia coli was optimized by applying a systematic approach. Firstly, a complete biosynthetic process to α-pinene was constructed by bioprospecting the key enzymes pinene synthase (PtPS1Q457L) and geranyl diphosphate synthase (AgGPPS). Subsequently, the appropriate N-terminal truncated sites of pinene synthase were determined by analyzing the hydrophilicity/hydrophobicity of PtPS1Q457L. The accumulation of extracellular α-pinene was further increased 2.5 times by tailoring an appropriate length of inactive terminal of pinene synthase. Fine-tuning of IPTG concentration and induction point led to the synthesis of α-pinene at the yield of 541.8 mg/L at the shake flask level. Finally, further optimization of the fed-batch fermentation process enabled a α-pinene titer of 1035.1 mg/L in a 1.3 L bioreactor, which is the highest according to currently available reports.