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

Abstract

Background β-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. Results A 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. Conclusion A 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.

Enhancement of β-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of β-Caryophyllene Synthase and Engineering the Chassis

“…Phytoextraction is presently the main production route of β-caryophyllene; thus, β-caryophyllene synthases from various plants have been getting attention previously. ,− Among them, β-caryophyllene synthase of A. annua was widely used for heterologous β-caryophyllene synthesis in different chassis cells due to the relatively high level of β-caryophyllene compared with other plants. ,,,, With the development of genome data mining in recent years, other genes encoding β-caryophyllene synthase were reported, such as those from plant endophyte Cladosporium sp. and Folsomia candida .…”

“…Currently, β-caryophyllene is obtained mainly from plants; however, this possesses disadvantages such as low abundance and complex extraction processes. The production of β-caryophyllene from renewable feedstocks by microbial cell factories is regarded as a promising and potentially sustainable alternative. − …”

Biokatalytische Ganzzellproduktion des Sesquiterpens Presilphiperfolan‐8β‐ol in stoffwechseloptimierten Escherichia coli

“…Mit den Erkenntnissen des ,,metabolic engineering'' ko ¨nnen so Stoffwechselwege fu ¨r verschiedenste Produkte entwickelt werden. In fru ¨heren Arbeiten hat sich die Ganzzellproduktion von verschiedenen Sesquiterpenen, wie (-)-Patchoulol [18], β-Caryophyllen [19], α-Humulen [20] und (-)-α-Bisabolol [21], bereits als mo ¨glich gezeigt [22]. Um eine ganzzellkatalytische Produktion von Sesquiterpenen zu erreichen, muss eine zellula ¨re Produktion des Vorla ¨ufers FPP als Substrat fu ¨r die STC sichergestellt sein.…”

AabHLH112, a bHLH transcription factor, positively regulates sesquiterpenes biosynthesis in Artemisia annua

“…Artemisia annua, a traditional Chinese medicinal plant, not only produces the well-known artemisinin, but also produces a number of sesquiterpenes that play an important role in plants' stress resistance, communication, and growth regulation (Chadwick et al, 2013). β-caryophyllene, epi-cedrol, and βfarnesene, three kinds of sesquiterpenes, are used as spice in the food industry and biological pesticides in agricultural production Cheng et al, 2022). Besides, they also have a significant value in the pharmaceutical industry (Cheng et al, 2022).…”

“…β-caryophyllene, epi-cedrol, and βfarnesene, three kinds of sesquiterpenes, are used as spice in the food industry and biological pesticides in agricultural production Cheng et al, 2022). Besides, they also have a significant value in the pharmaceutical industry (Cheng et al, 2022). β-Caryophyllene was found to has good curative effect on colitis (Bento et al, 2011), cerebral ischemia (Chang et al, 2013), diabetes (Basha and Sankaranarayanan, 2014), anxiety and depression (Bahi et al, 2014), liver fibrosis (Mahmoud et al, 2014), and osteoarthritis (Rufino et al, 2015).…”