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Background Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. Results This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. Conclusions This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Yarrow ( Achillea millefolium  L.) is an important medicinal and aromatic plant the active biologically compounds in particular essential oil is used as raw material in the pharmaceutical, food, and cosmetics industries. The impacts of the foliar spraying of chitosan (control and 2.5 g/L chitosan), the use of soil-based biochar (control and soil application at 2.0 kg/m 2 ) under three moisture levels (irrigation at 80–85%, 60–65%, and 40–45% F.C.) on the agronomic traits and essential oil of yarrow were investigated. Results indicated that the deficit irrigation meaningfully decreased the yields of biological and flower, and essential oil yield, however, the contents of proline and essential oil under reduced irrigation significantly increased. According to essential oils analysis by GC-FID and GC/MS, the major constituents were α-pinene, 1,8-cineole, borneol, β-bisabolene, and caryophyllene oxide. A considerable increase and decrease were observed respectively in the monoterpenes and sesquiterpenes contents under reduced irrigation. The utilization of biochar along chitosan maintained higher secondary metabolites in particular α–pinene, β-myrcene, borneol, and 1,8-cineole under deficit irrigation. Generally, it appears that using the foliar spraying of chitosan along the soil application of biochar can be effective in improving the qualitative and quantitative features of the essential oil of A. millefolium in arid and semiarid climates.

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.

Summary The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non‐economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof‐of‐principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α‐bisabolene, β‐bisabolene and γ‐bisabolene through heterologous expression of the α‐bisabolene synthase from Abies grandis, the β‐bisabolene synthase gene from Zingiber officinale and the γ‐bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher‐level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products. To our knowledge, we have unprecedentedly verified the function of α‐bisabolene synthase, β‐bisabolene synthase and γ‐bisabolene synthase in Y. lipolytica and successfully employed the enzyme to synthesize bisabolene in Y. lipolytica for the first time. Our study reports for the first time the utilization of terpenoid efflux pumps to improve the secretion, and thus the production, of bisabolene in microbial cell factories. We demonstrated for the first time the conversion of waste cooking oil to bisabolene, which has important potential applications in waste management as well as economical and sustainable production of valuable bisabolene from waste feedstocks.

Secondary organic aerosol (SOA) is an important constituent of the atmosphere where SOA particles are formed chiefly by the condensation or reactive uptake of oxidation products of volatile organic compounds (VOCs). The mass yield in SOA particle formation, as well as the chemical composition and volatility of the particles, is determined by the identity of the VOC precursor(s) and the oxidation conditions they experience. In this study, we used an oxidation flow reactor to generate biogenic SOA from the oxidation of Scots pine emissions. Mass yields, chemical composition and volatility of the SOA particles were characterized and compared with SOA particles formed from oxidation of α-pinene and from a mixture of acyclic–monocyclic sesquiterpenes (farnesenes and bisabolenes), which are significant components of the Scots pine emissions. SOA mass yields for Scots pine emissions dominated by farnesenes were lower than for α-pinene but higher than for the artificial mixture of farnesenes and bisabolenes. The reduction in the SOA yield in the farnesene- and bisabolene-dominated mixtures is due to exocyclic C=C bond scission in these acyclic–monocyclic sesquiterpenes during ozonolysis leading to smaller and generally more volatile products. SOA particles from the oxidation of Scots pine emissions had similar or lower volatility than SOA particles formed from either a single precursor or a simple mixture of VOCs. Applying physical stress to the Scots pine plants increased their monoterpene, especially monocyclic β-phellandrene, emissions, which further decreased SOA particle volatility and increased SOA mass yield. Our results highlight the need to account for the chemical complexity and structure of real-world biogenic VOC emissions and stress-induced changes to plant emissions when modelling SOA production and properties in the atmosphere. These results emphasize that a simple increase or decrease in relative monoterpene and sesquiterpene emissions should not be used as an indicator of SOA particle volatility.

Background:Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts.Results:In this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to havesuperior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel bio-compatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars.Conclusions:This study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes.

Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.

Gongora is a neotropical epiphytic orchid found from Mexico to South America, with 11 species recorded in Brazil. Little is known about the aromas produced by the flowers of these orchid species. This study aimed to identify the volatile constituents of the flowers of Gongora histrionica (1), G. jauariensis (1), G. longiracemosa (2), G. minax (1), and G. pleiochroma (2), all of which are found in the Amazon. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the volatile constituents of Gongora. Additionally, multivariate statistical analysis was employed to evaluate the variability of volatile constituents in their floral aromas. The principal constituents (>25%) of Gongora specimens were (Z)-α-bisabolene, terpinen-4-ol, cis-β-elemenone, (E,E)-geranyl linalool, β-bisabolene, (E,E)-α-farnesene, and 1,8-cineole. Based on the identified compounds, the multivariate statistical analysis revealed seven distinct groups for the Gongora volatile concentrates, indicating a significant variability between the studied species. These results contribute to a better understanding of the genus Gongora chemotaxonomy analysis.

The eco-friendly management of mosquitoes with novel and effective larvicides and oviposition deterrents is a crucial challenge to prevent outbreaks of mosquito-borne diseases. However, most of the herbal formulations tested in these years showed LC 50 values higher of 40 ppm, and significant oviposition deterrent activity only when tested at relatively higher doses (> 50 μg/ml). Herein, we studied the chemical composition of the Galinsoga parviflora essential oil (EO). This plant is an annual herb native to South America naturalized all over the world. We tested the EO larvicidal and oviposition deterrent action on 6 mosquito species. Totally 37 compounds were identified in the EO of G. parviflora by GC and GC-MS analyses. The major constituent was ( Z )- γ -bisabolene (38.9%). The G. parviflora EO and ( Z )- γ -bisabolene showed acute toxicity on An. stephensi ( LC 50  = 31.04 and 2.04 μg/ml, respectively), Ae. aegypti (LC 50  = 34.22 and 2.26 μg/ml, respectively), Cx . quinquefasciatus (LC 50  = 37.10 and 2.47 μg/ml, respectively), An. subpictus (LC 50  = 40.97 and 4.09 μg/ml, respectively), Ae. albopictus (LC 50  = 45.55 and 4.50 μg/ml, respectively) and Cx. tritaeniorhynchus ( LC 50  = 49.56 and 4.87 μg/ml, respectively) larvae. Furthermore, the oviposition deterrent potential of the G. parviflora EO and ( Z )- γ -bisabolene was studied on six mosquito vectors, showing that 25 μg/ml of ( Z )- γ -bisabolene led to an Oviposition Activity Index lower of − 0.79 in all tested mosquito vectors. Overall, all larvicidal LC 50 values estimated for ( Z )- γ -bisabolene were lower than 5 μg/ml. This result far encompasses current evidences of toxicity reported for the large majority of botanical products currently tested against mosquito young instars, allowing us to propose this compound as an highly effective mosquito larvicide and oviposition deterrent.

Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. Operating in a two-step semi-batch mode over a period of eight days, volumetric yields of ( E )-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L −1 . Likewise, yields of the sesquiterpene alcohols (−)-patchoulol and (−)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L −1 and 96.3 ± 2.2 mg * L −1 , respectively. While specific productivity was compromised particularly for ( E )-α-bisabolene in the HDC system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for ( E )-α-bisabolene than for the hydroxylated terpenoids. Together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria.