Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool

Abstract

Background: Geranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis.

Results: We first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far.

Conclusions: Efficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

Keywords: Escherichia coli; Biosynthesis; Geranyllinalool; Isopentenol utilization pathway.

Fig. 1

Development of a biosynthetic pathway for geranyllinalool production

Fig. 2

Geranyllinalool synthases screened at an induction temperature of 25℃ with 0.5 mM IPTG for geranyllinalool biosynthesis enhancement. Asterisk denotes statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3

Optimization of fermentation conditions for geranyllinalool production by E. coli GL01. (A) Geranyllinalool distribution within the cellular milieu assessed following induction at 25℃ with 0.5 mM IPTG. (B) Effects of various substrates on geranyllinalool production at an induction temperature of 25℃ with 0.5 mM IPTG. (C) Influence of different concentrations of prenol on geranyllinalool production was examined under induction conditions at 25℃ with 0.5 mM IPTG. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Optimization of induction conditions for geranyllinalool production by E coli GL01. (A) Effects of various carbon sources on geranyllinalool production with an induction temperature of 25℃ and 0.5 mM IPTG. (B) Effects of IPTG temperature on geranyllinalool production with 10 g/L initial glycerol and 0.5 mM IPTG. (C) Effects of IPTG concentration on geranyllinalool production with 10 g/L initial glycerol and induction at 20℃. Asterisk denotes statistically significant differences: *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

Fed-batch fermentation of recombinant E. coli GL01 in a 5-L bioreactor for geranyllinalool production (A) and by-product formation (B) with glycerol as the carbon source