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Background The sesquiterpene germacrene A is a direct precursor of ß-elemene that is a major component of the Chinese medicinal herb Curcuma wenyujin with prominent antitumor activity. The microbial platform for germacrene A production was previously established in Saccharomyces cerevisiae using the germacrene A synthase (LTC2) of Lactuca sativa . Results We evaluated the performance of LTC2 ( Ls GAS) as well as nine other identified or putative germacrene A synthases from different sources for the production of germacrene A. Av GAS, a synthase of Anabaena variabilis , was found to be the most efficient in germacrene A production in yeast. Av GAS expression alone in S. cerevisiae CEN.PK2-1D already resulted in a substantial production of germacrene A while LTC2 expression did not. Further metabolic engineering the yeast using known strategies including overexpression of tHMGR1 and repression of squalene synthesis pathway led to an 11-fold increase in germacrene A production. Site-directed mutagenesis of Av GAS revealed that while changes of several residues located within the active site cavity severely compromised germacrene A production, substitution of Phe23 located on the lateral surface with tryptophan or valine led to a 35.2% and 21.8% increase in germacrene A production, respectively. Finally, the highest production titer of germacrene A reached 309.8 mg/L in shake-flask batch culture. Conclusions Our study highlights the potential of applying bacterial sesquiterpene synthases with improved performance by mutagenesis engineering in producing germacrene A.

This study aimed to investigate the chemical composition of the leaf essential oil from Ivoirian Isolona dewevrei. A combination of chromatographic and spectroscopic techniques (GC(RI), GC-MS and 13C-NMR) was used to analyze two oil samples (S1 and S2). Detailed analysis by repetitive column chromatography (CC) of essential oil sample S2 was performed, leading to the isolation of four compounds. Their structures were elucidated by QTOF-MS, 1D and 2D-NMR as (10βH)-1β,8β-oxido-cadin-4-ene (38), 4-methylene-(7αH)-germacra-1(10),5-dien-8β-ol (cis-germacrene D-8-ol) (52), 4-methylene-(7αH)-germacra-1(10),5-dien-8α-ol (trans-germacrene D-8-ol) (53) and cadina-1(10),4-dien-8β-ol (56). Compounds 38, 52 and 53 are new, whereas NMR data of 56 are reported for the first time. Lastly, 57 constituents accounting for 95.5% (S1) and 97.1% (S2) of the whole compositions were identified. Samples S1 and S2 were dominated by germacrene D (23.6 and 20.5%, respectively), followed by germacrene D-8-one (8.9 and 8.7%), (10βH)-1β,8β-oxido-cadin-4-ene (7.3 and 8.7), 4-methylene-(7αH)-germacra-1(10),5-dien-8β-ol (7.8 and 7.4%) and cadina-1(10),4-dien-8β-ol (7.6 and 7.2%). Leaves from I. dewevrei produced sesquiterpene-rich essential oil with an original chemical composition, involving various compounds reported for the first time among the main components. Integrated analysis by GC(RI), GC-MS and 13C-NMR appeared fruitful for the knowledge of such a complex essential oil.

Germacrene A (GA) is a key intermediate for the synthesis of medicinal active compounds, especially for β-elemene, which is a broad-spectrum anticancer drug. The production of sufficient GA in the microbial platform is vital for the precursors supply of active compounds. In this study, Escherichia coli BL21 Star (DE3) was used as the host and cultivated in SBMSN medium, obtaining a highest yield of FPP. The GA synthase from Lactuca sativa (LTC2) exhibited the highest level of GA production. Secondly, two residues involved in product release (T410 and T392) were substituted with Ser and Ala, respectively, responsible for relatively higher activities. Next, substitution of selected residues S243 with Asn caused an increase in activity. Furthermore, I364K-T410S and T392A-T410S were created by combination with the beneficial mutation, and they demonstrated dramatically enhanced titers with 1.90-fold and per-cell productivity with 5.44-fold, respectively. Finally, the production titer of GA reached 126.4 mg/L, and the highest productivity was 7.02 mg/L.h by the I364K-T410S mutant in a shake-flask batch culture after fermentation for 18 h. To our knowledge, the productivity of the I364K-T410S mutant is the highest level ever reported. These results highlight a promising method for the industrial production of GA in E. coli , and lay a foundation for pathway reconstruction and the production of valuable natural sesquiterpenes.

Germacranes are important intermediates in the biosynthesis of eudesmane and guaiane sesquiterpenes. After their initial formation from farnesyl diphosphate, these neutral intermediates can become reprotonated for a second cyclisation to reach the bicyclic eudesmane and guaiane skeletons. This review summarises the accumulated knowledge on eudesmane and guaiane sesquiterpene hydrocarbons and alcohols that potentially arise from the achiral sesquiterpene hydrocarbon germacrene B. Not only compounds isolated from natural sources, but also synthetic compounds are dicussed, with the aim to give a rationale for the structural assignment for each compound. A total number of 64 compounds is presented, with 131 cited references.

β-elemene is an effective anti-cancer component which has been widely used in clinic. However, it still relies on the extraction from the Chinese medicine plant Curcuma wenyujin, which seriously limits its application. Synthetic biology offers a promising approach to satisfy its supply. β-elemene is derived from germacrene A (GA), which is synthesized by germacrene A synthase (GAS), through Cope rearrangement under heat condition instead of enzymatic reaction. In this study, an effective germacrene A synthase (ScGAS) was identified from Solidago canadensis which could produce GA when expressed in E.coli. By introducing the heterogeneous MVA pathway to enrich the FPP pool, the strain yielded 147 mg/L of GA in shake flasks which represented 2.98-fold improvement over the initial one. Moreover, combining molecular docking with phylogeny analysis of ScGAS largely narrowed down the category of its key residues' mutagenesis. The Y376L mutant showed the highest yield of 487 mg/L which was almost 10-fold higher than the initial yield. These results indicate that diverting the metabolism of the host and enzyme mutagenesis based on the combination of molecular docking and phylogeny analysis are of great value to constructing terpenoids chassis.

Germacrene D, a sesquiterpenoid compound found mainly in plant essential oils at a low level as (+) and/or (−) enantiomeric forms, is an ingredient for the fragrance industry, but a process for the sustainable supply of enantiopure germacrene D is not yet established. Here, we demonstrate metabolic engineering in yeast ( Saccharomyces cerevisiae ) achieving biosynthesis of enantiopure germacrene D at a high titer. To boost farnesyl pyrophosphate (FPP) flux for high-level germacrene D biosynthesis, a background yeast chassis (CENses5C) was developed by genomic integration of the expression cassettes for eight ergosterol pathway enzymes that sequentially converted acetyl-CoA to FPP and by replacing squalene synthase promoter with a copper-repressible promoter, which restricted FPP flux to the competing pathway. Galactose-induced expression of codon-optimized plant germacrene D synthases led to 13–30 fold higher titers of (+) or (−)-germacrene D in CENses5C than the parent strain CEN.PK2.1C. Furthermore, genomic integration of germacrene D synthases in GAL80 , LPP1 and rDNA loci generated CENses8(+D) and CENses8(−D) strains, which produced 41.36 µg/ml and 728.87 µg/ml of (+) and (−)-germacrene D, respectively, without galactose supplementation. Moreover, coupling of mitochondrial citrate pool to the cytosolic acetyl-CoA, by expressing a codon-optimized ATP-citrate lyase of oleaginous yeast, resulted in 137.71 µg/ml and 815.81 µg/ml of (+) or (−)-germacrene D in CENses8(+D)* and CENses8(−D)* strains, which were 67–120 fold higher titers than in CEN.PK2.1C. In fed-batch fermentation, CENses8(+D)* and CENses8(−D)* produced 290.28 µg/ml and 2519.46 µg/ml (+) and (−)-germacrene D, respectively, the highest titers in shake-flask fermentation achieved so far. Key points • Engineered S. cerevisiae produced enantiopure (+) and (−)-germacrene D at high titers • Engineered strain produced up to 120-fold higher germacrene D than the parental strain • Highest titers of enantiopure (+) and (−)-germacrene D achieved so far in shake-flask

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low‐calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS‐short gene and not the CiGAS‐long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction.

Main conclusionThe study demonstrated thatArtemisia pallensroots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene A contributing to major root volatiles.Davana (Artemisia pallens Bess) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatile composition, and the genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields ~ 0.05% of EO, which is primarily composed of sesquiterpenes β-elemene, neryl isovalerate, β-selinene, and α-selinene, and trace amounts of monoterpenes β-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of β-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Detailed analysis of assay products revealed that β-elemene was the thermal rearrangement product of germacrene A. The functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in roots, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of germacrene A. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile.

β-elemene is an effective anti-cancer component which has been widely used in clinic. However, it still relies on the extraction from the Chinese medicine plant , which seriously limits its application. Synthetic biology offers a promising approach to satisfy its supply. β-elemene is derived from germacrene A (GA), which is synthesized by germacrene A synthase (GAS), through Cope rearrangement under heat condition instead of enzymatic reaction. In this study, an effective germacrene A synthase (ScGAS) was identified from which could produce GA when expressed in . By introducing the heterogeneous MVA pathway to enrich the FPP pool, the strain yielded 147 mg/L of GA in shake flasks which represented 2.98-fold improvement over the initial one. Moreover, combining molecular docking with phylogeny analysis of ScGAS largely narrowed down the category of its key residues' mutagenesis. The Y376L mutant showed the highest yield of 487 mg/L which was almost 10-fold higher than the initial yield. These results indicate that diverting the metabolism of the host and enzyme mutagenesis based on the combination of molecular docking and phylogeny analysis are of great value to constructing terpenoids chassis.

Background The sesquiterpene germacrene D is a highly promising product due to its wide variety of insecticidal activities and ability to serve as a precursor for many other sesquiterpenes. Biosynthesis of high value compounds through genome mining for synthases and metabolic engineering of microbial factories, especially Saccharomyces cerevisiae , has been proven to be an effective strategy. However, there have been no studies on the de novo synthesis of germacrene D from carbon sources in microbes. Hence, the construction of the S. cerevisiae cell factory to achieve high production of germacrene D is highly desirable. Results We identified five putative sesquiterpene synthases (AcTPS1 to AcTPS5) from Acremonium chrysogenum and the major product of AcTPS1 characterized by in vivo, in vitro reaction and NMR detection was revealed to be (–)-germacrene D. After systematically comparing twenty-one germacrene D synthases, AcTPS1 was found to generate the highest amount of (–)-germacrene D and was integrated into the terpene precursor-enhancing yeast strain, achieving 376.2 mg/L of (–)-germacrene D. Iterative engineering was performed to improve the production of (–)-germacrene D, including increasing the copy numbers of AcTPS1 , tHMG1 and ERG20 , and downregulating or knocking out other inhibitory factors (such as erg9 , rox1 , dpp1 ). Finally, the optimal strain LSc81 achieved 1.94 g/L (–)-germacrene D in shake-flask fermentation and 7.9 g/L (–)-germacrene D in a 5-L bioreactor, which is the highest reported (–)-germacrene D titer achieved to date. Conclusion We successfully achieved high production of (–)-germacrene D in S. cerevisiae through terpene synthase mining and metabolic engineering, providing an impressive example of microbial overproduction of high-value compounds.