Enhanced (−)-α-Bisabolol Productivity by Efficient Conversion of Mevalonate in Escherichia coli

Abstract

(−)-α-Bisabolol, a naturally occurring sesquiterpene alcohol, has been used in pharmaceuticals and cosmetics owing to its beneficial effects on inflammation and skin healing. Previously, we reported the high production of (−)-α-bisabolol by fed-batch fermentation using engineered Escherichia coli (E. coli) expressing the exogenous mevalonate (MVA) pathway genes. The productivity of (−)-α-bisabolol must be improved before industrial application. Here, we report enhancement of initial (−)-α-bisabolol productivity to 3-fold higher than that observed in our previous study. We first harnessed a farnesyl pyrophosphate (FPP)-resistant mevalonate kinase 1 (MvaK1) from an archaeon Methanosarcina mazei (M. mazei) to create a more efficient heterologous MVA pathway that produces (−)-α-bisabolol in the engineered E. coli. The resulting strain produced 1.7-fold higher (−)-α-bisabolol relative to the strain expressing a feedback-inhibitory MvaK1 from Staphylococcus aureus (S. aureus). Next, to efficiently convert accumulated MVA to (−)-α-bisabolol, we additionally overexpressed genes involved in the lower MVA mevalonate pathway in E. coli containing the entire MVA pathway genes. (−)-α-Bisabolol production increased by 1.8-fold with reduction of MVA accumulation, relative to the control strain. Finally, we optimized the fermentation conditions including inducer concentration, aeration and enzymatic cofactor. The strain was able to produce 8.5 g/L of (−)-α-bisabolol with an initial productivity of 0.12 g/L h in the optimal fed-batch fermentation. Thus, the microbial production of (−)-α-bisabolol would be an economically viable bioprocess for its industrial application.

Characterization of γ-Cadinene Enzymes in Ganoderma lucidum and Ganoderma sinensis from Basidiomycetes Provides Insight into the Identification of Terpenoid Synthases

“…In addition, as one of the four stereoisomers of α-bisabolol, (+)-α-bisabolol, 40 (−)-α-bisabolol, 28 , 41 − 44 (+)- epi -α-bisabolol, 45 and (−)- epi -α-bisabolol, 46 (−)-α-bisabolol, which is a monocyclic sesquiterpene alcohol, is highly valued and widely used as an active ingredient in the cosmetics and pharmaceutical industries. 28 , 47 The productivity of (−)-α-bisabolol has been improved by using engineered microbes and high-efficiency BOS for the industrial production.…”

(−)-α-Bisabolol Production in Engineered Escherichia coli Expressing a Novel (−)-α-Bisabolol Synthase from the Globe Artichoke Cynara cardunculus var. Scolymus

“…The resulting plasmids were used for purification of BOS enzymes. To generate a pTSN-CcBisa-Mm plasmid, a pTSN-Bisa-Mm plasmid 13 was digested with the MluI and AatII restriction enzymes to remove MrBOS (V1). Three DNA fragments (F1, F2, and F3) were amplified by PCR using pTSN-Bisa-Mm, E. coli codon-optimized CcBOS, pTSN-Bisa-Mm, and the corresponding primers (Table S1).…”

“…12 (−)-α-Bisabolol productivity was ameliorated by reinforcing MVA flux to FPP and replacing MVA kinase 1 (MvaK1) with MvaK1 from Methanosarcina mazei to resist FPP feedback inhibition. 13,14 Terpene synthase (TPS) plays an essential role in bacterial terpenoid production, as most TPSs have broad substrate specificity and promiscuous activity to regulate terpenoid diversity. 15 TPS from Medicago truncatula and Abies grandis synthesized 27 and 52 products, respectively.…”

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“… 72–75 The highest isoprene titres achieved so far use the feedback-resistant Methanosarcina mazei M5K; 76 (−)-α-bisabolol titres were increased 1.7-fold, reaching almost 600 mg L −1 by replacing S. aureus M5K with the M. mazei homolog. 73 Protein engineering can also be used to improve substrate affinity, and thermal and pH stabilities of feedback-inhibited M5K. An E. coli strain harbouring the S. cerevisiae M5K V13D/S148I/V301E variant produced 1.43 g L −1 lycopene, a titre that is 2.4-fold higher compared to the strain containing the wild-type homolog.…”