Highly efficient biosynthesis of β-caryophyllene with a new sesquiterpene synthase from tobacco.

backgroundβ-Caryophyllene, a kind of bicyclic sesquiterpene, is mainly used as a spice in the food and cosmetic industries. Furthermore, it also has significant value in the pharmaceutical industry and is now considered to be used as a new fuel. As a chemical energy heterotrophic microorganism, Escherichia coli can produce a large amount of acetyl-CoA through aerobic respiration, and acetyl-CoA is the common precursor substance in the biosynthesis of all terpenoids. Therefore, E. coli has the potential to be a cell factory to produce terpenoids.

resultsA new gene of β-caryophyllene synthase (TPS7) was found by analyzing the genome of Nicotiana tabacum L. using bioinformatics methods. The gene was overexpressed in engineered E. coli with a heterogeneous mevalonate (MVA) pathway to build a recombinant strain CAR1. Subsequent cultivation experiments in shake flask of engineered strain CAR1 verified that 16.1 mg/L β-caryophyllene was detected from the fermentation broth in the shake flask after induction for 24 h with IPTG. The toxic by-product of farnesyl acetate was detected during the process, and CAR1 showed a heavily cellular accumulation of product. We constructed an engineered strain CAR2, in which the downstream genes of the MVA pathway were integrated into the E. coli chromosome, successfully increasing β-caryophyllene production to 100.3 mg/L. The highest production of β-caryophyllene during the fed-batch fermentation was 4319 mg/L. Then we employed in situ extraction fermentation to successfully increase the production of β-caryophyllene by 20% to 5142 mg/L.

conclusionA new sesquiterpene synthase, TPS7, from tobacco was found to be able to produce β-caryophyllene with high efficiency. Based on this, an engineered E. coli was constructed to produce a much higher concentration of β-caryophyllene than the previous studies. During the fermentation process, we observed that β-caryophyllene tends to accumulate in intracellular space, which will eventually influence the activity of engineered E. coli. As a result, we solved this by metabolism regulation and in situ extractive fermentation.