Geraniol Synthase Research Articles

Engineering a Carotenoid-Overproducing Strain of Azospirillum brasilense for Heterologous Production of Geraniol and Amorphadiene.

Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Engineering Corynebacterium glutamicum for Geraniol Production

Geraniol is a monoterpenoid alcohol with various applications in food, cosmetics, and healthcare. Corynebacterium glutamicum is a potential platform for terpenoids production because it harbors the methylerythritol phosphate pathway. To engineer C. glutamicum to produce geraniol, two different truncated geraniol synthases (GESs) were respectively expressed, and strain LX02 expressing the truncated GESs from Valeriana officinalis (tVoGES) produced 0.3 mg/L of geraniol. Then, three geranyl diphosphate synthases (GPPSs) were combinatorially co-expressed with tVoGES to improve geraniol production. The amounts of produced geraniol were all higher than that produced by strain LX02. Strain LX03 co-expressing ERG20F96W–N127W (ERG20WW) and tVoGES produced the highest amount, 5.4 mg/L. Subsequently, the co-overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) further increased the production to 12.2 mg/L in strain LX03. Lastly, the production of geraniol was increased to 15.2 mg/L via fermentation optimization. To our knowledge, this is the first report on the engineering of C. glutamicum to produce geraniol and thus can serve as a reference for other monoterpenoid production studies.

Characterization of Terpene Synthase from Tea Green Leafhopper Being Involved in Formation of Geraniol in Tea (Camellia sinensis) Leaves and Potential Effect of Geraniol on Insect-Derived Endobacteria.

When insects attack plants, insect-derived elicitors and mechanical damage induce the formation and emission of plant volatiles that have important ecological functions and flavor properties. These events have mainly been studied in model plants, rather than crop plants. Our study showed that tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda), a major pest infesting tea attack significantly induced the emission of geraniol from tea leaves, but did not affect the crude enzyme activity of geraniol synthase in tea leaves. An enzyme extract of E. (M.) onukii specifically produced geraniol from geraniol diphosphate. Furthermore, a terpene synthase (EoTPS) was isolated from E. (M.) onukii. This terpene synthase was able to convert geraniol diphosphate to geraniol in vitro. In addition, geraniol had in vitro ability to inhibit the growth of Acinetobacter johnsonii that is endobacterial isolated from E. (M.) onukii. This information illustrates that elicitors from piercing-sucking insects can induce the formation of volatiles from crop plants and advances our understanding of the roles of plant volatiles in the interaction among crops-insects-microorganisms.

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

BackgroundGeraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds—which are often utilised for anti-microbial and anti-fungal functions in their host plant.ResultsIn this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed.ConclusionIn this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.

Promoter and methylation status analysis revealed the importance of PkGES gene in picroside biosynthesis in Picrorhiza kurrooa

Picrorhiza kurrooa Royle ex Benth., an important high altitude plant produces picrosides, the monoterpenoids of high therapeutic value. Production of geraniol in the iridoid branch of the picroside biosynthetic pathway is a key step in the biosynthesis of picrosides. Geraniol synthase, catalyzes the conversion of geranyl pyrophosphate into geraniol. In view of this, here we report the identification and sequence analysis of PkGES promoter. The PkGES promoter sequence was isolated following a directional genome walking approach. Presence of similar cis elements such as GT1 consensus elements, LTREs, DREs, WRKY binding elements, PREATPRODH motif, BIHD1OS motif, SEBFCONSSTPR10A motif and MeJA responsive elements in the promoter sequence of PkGES documented its multiple stress responsive characteristic signifying its role in adaptation. Further, PkGES showed differential expression pattern in leaves, rhizome and roots. To elucidate whether the differential expression pattern of PkGES correlates with its epigenetic regulation, DNA methylation analysis of promoter as well as coding region of PkGES was conducted. The DNA methylation levels in the promoter sequence of PkGES gene were found to be dominated at CG followed by CHH and CHG sequence contexts. Epigenetic regulation (DNA methylation) of promoter as well as coding region of PkGES suggested its role in the regulation of PkGES transcripts in diverse tissues. The present study has revealed the presence of several stress inducible cis elements in PkGES promoter sequence and its tissue specific epigenetic regulation in P. kurrooa.

Transcriptome analysis and targeted metabolic profiling for pathway elucidation and identification of a geraniol synthase involved in iridoid biosynthesis from Gardenia jasminoides

Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

Overexpression of geraniol synthase induces heat stress susceptibility in Nicotiana tabacum.

Transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase exhibit hypersensitivity to thermal stress, possibly due to suppressed sugar metabolism and transcriptional regulation of genes involved in thermal stress tolerance. Monoterpene alcohols function in plant survival strategies, but they may cause self-toxicity to plants due to their hydrophobic and highly reactive properties. To explore the role of these compounds in plant stress responses, we assessed transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase (GES plants). Growth, morphology and photosynthetic efficiency of GES plants were not significantly different from those of control plants (wild-type and GUS-transformed plants). While GES plants' direct defenses against herbivores or pathogens were similar to those of control plants, their indirect defense (i.e., attracting herbivore enemy Nesidiocoris tenuis) was stronger compared to that of control plants. However, GES plants were susceptible to cold stress and even more susceptible to extreme heat stress (50°C), as shown by decreased levels of sugar metabolites, invertase activity and its products (Glc and Fru), and leaf starch granules. Moreover, GES plants showed decreased transcription levels of the WRKY33 transcription factor gene and an aquaporin gene (PIP2). The results of this study show that GES plants exhibit enhanced indirect defense ability against herbivores, but conversely, GES plants exhibit hypersensitivity to heat stress due to suppressed sugar metabolism and gene regulation for thermal stress tolerance.

Functional Analysis of Four Terpene Synthases in Rose-Scented Pelargonium Cultivars (Pelargonium × hybridum) and Evolution of Scent in the Pelargonium Genus.

Pelargonium genus contains about 280 species among which at least 30 species are odorant. Aromas produced by scented species are remarkably diverse such as rose, mint, lemon, nutmeg, ginger and many others scents. Amongst odorant species, rose-scented pelargoniums, also named pelargonium rosat, are the most famous hybrids for their production of essential oil (EO), widely used by perfume and cosmetic industries. Although EO composition has been extensively studied, the underlying biosynthetic pathways and their regulation, most notably of terpenes, are largely unknown. To gain a better understanding of the terpene metabolic pathways in pelargonium rosat, we generated a transcriptome dataset of pelargonium leaf and used a candidate gene approach to functionally characterise four terpene synthases (TPSs), including a geraniol synthase, a key enzyme responsible for the biosynthesis of the main rose-scented terpenes. We also report for the first time the characterisation of a novel sesquiterpene synthase catalysing the biosynthesis of 10-epi-γ-eudesmol. We found a strong correlation between expression of the four genes encoding the respective TPSs and accumulation of the corresponding products in several pelargonium cultivars and species. Finally, using publically available RNA-Seq data and de novo transcriptome assemblies, we inferred a maximum likelihood phylogeny from 270 pelargonium TPSs, including the four newly discovered enzymes, providing clues about TPS evolution in the Pelargonium genus. Notably, we show that, by contrast to other TPSs, geraniol synthases from the TPS-g subfamily conserved their molecular function throughout evolution.

The impact of cold stress on genes expression pattern of mono- and sesquiterpene biosynthesis and their contents in Ocimum basilicum L.

Basil (Ocimum basilicum L.) contains valuable monoterpene and sesquiterpene compounds used for medical purposes. Environmental stresses are suggested to change the essential oil composition in medicinal plants. In the current investigation, an experiment was arranged in greenhouse to study the effect of cold stress on genes expression patterns of linalool synthase (LIS), β-myrcene synthase (MYS), γ-cadinene synthase (CDS), germacrene D synthase (GDS) and geraniol synthase (GES) which is involved in monoterpenes and sesquiterpenes biosynthesis in O. basilicum. The monoterpenes and sesquiterpenes composition and content were also investigated. Plants were exposed to temperatures 22 (control), 4 and 10 °C at 6 to 8 leaf stage for 12, 24 and 48 h. The genes expression levels were determined by real time PCR in plant leaves. Essential oil was extracted at flowering stage by distillation using Clevenger apparatus and its compounds were identified using gas chromatography/mass spectrometry (GC-MS). The results revealed that the LIS expression increasingly occurred to 4.86 fold at 10 °C after 12 h while that of GES reached to 5.7 fold at 10 °C after 48 h. Temperature 4 °C for 12 h increased the expression levels of MYS and GDS genes to 41.5 and 14.2 fold, respectively, while the expression level of CDS increased to 25.5 fold at 4 °C for 48 h. Significant differences (P ≤ 0.01) were observed among treatments with respect to all compounds except α-pinene and camphene. The maximum proportion of geraniol and γ-cadinene were observed at 4 °C for 24 h, while the maximum proportion of germacrene D and α-bergamotene obtained at 10 °C for 12 h. The highest proportion of 1, 8-cineole was achieved at 4 °C for 48 h. Positive associations between germacrene D content and GDS expression (r = 0.8, P ≤ 0.05) and γ-cadinene and CDS expression (r = 0.78, P ≤ 0.05) proposed that the content of terpenoid compounds in basil can be enhanced through increasing the expression levels of genes involved in their biosynthesis.

An enzyme-coupled assay enables rapid protein engineering for geraniol production in yeast

Geraniol is an important monoterpene alcohol with various industrial applications. The biological synthesis of geraniol requires the activity of geraniol synthase (GES). Despite several engineering efforts to improve catalytic rates of GES, overall efforts have been limited by the lack of a high-throughput screen. Here, we developed a coupled enzyme-based fluorogenic assay that can detect geraniol as well as other medium to long chain alcohols (C4-C9). Aided by this rapid screening capability, we performed saturation mutagenesis of GES of Catharanthus roseus and identified a mutation of F418 to Q that improved production of geraniol. This robust screening assay enables more high-throughput analysis and engineering of geraniol and other alcohols in S. cerevisiae and E. coli.

Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus.

Catharanthus roseus is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in C. roseus. Here, to overcome this limitation and enhance MIAs levels in C. roseus, bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by Agrobacterium-mediated transformation. Both transient and stable overexpression of G(G)PPS and co-expression of G(G)PPS+GES significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic C. roseus plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in G(G)PPS but not in G(G)PPS+GES transgenic lines that correlated with transcript levels of peroxidase-1 (PRX1) involved in coupling of vindoline and catharanthine into 3′,4′-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T1) lines exhibited comparable transcript and metabolite levels to that of T0 lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in C. roseus at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in C. roseus through transgenic approaches.

Phylogenomic Mining of the Mints Reveals Multiple Mechanisms Contributing to the Evolution of Chemical Diversity in Lamiaceae

The evolution of chemical complexity has been a major driver of plant diversification, with novel compounds serving as key innovations. The species-rich mint family (Lamiaceae) produces an enormous variety of compounds that act as attractants and defense molecules in nature and are used widely by humans as flavor additives, fragrances, and anti-herbivory agents. To elucidate the mechanisms by which such diversity evolved, we combined leaf transcriptome data from 48 Lamiaceae species and four outgroups with a robust phylogeny and chemical analyses of three terpenoid classes (monoterpenes, sesquiterpenes, and iridoids) that share and compete for precursors. Our integrated chemical–genomic–phylogenetic approach revealed that: (1) gene family expansion rather than increased enzyme promiscuity of terpene synthases is correlated with mono- and sesquiterpene diversity; (2) differential expression of core genes within the iridoid biosynthetic pathway is associated with iridoid presence/absence; (3) generally, production of iridoids and canonical monoterpenes appears to be inversely correlated; and (4) iridoid biosynthesis is significantly associated with expression of geraniol synthase, which diverts metabolic flux away from canonical monoterpenes, suggesting that competition for common precursors can be a central control point in specialized metabolism. These results suggest that multiple mechanisms contributed to the evolution of chemodiversity in this economically important family.

Selectivity of geraniol synthase in aromatic species to control of cotton ramulosis

Ramulosis (Colletotrichum gossypii South var. cephalosporioides Costa) is a fungal disease of cotton (Gossypium hirsutum L.) that causes damage to leaves, stems, and bolls by reducing fiber production; it can be controlled by chemical fungicides. Geraniol is a monoterpenoid produced by some aromatic plant species whose fungicidal properties have been widely reported. Geraniol synthase (GES; EC 3.1.7.11) is the precursor enzyme involved in the biosynthetic chain of geraniol. Geraniol synthase (ges) gene transcripts were prospected in 11 aromatic species with molecular and phytopathological tools to identify promising accessions for further use in in vitro and in vivo assays involving the control of cotton ramulosis. Mentha pulegium L. oil highly expressed ges and inhibited fungal growth at 1000 uL L-1 in an in vitro assay. Validation assays were carried out in two environments and M. pulegium at 2000 uL L-1 reduced the initial and final severity indices of the disease to 48% and 52%, respectively, in preventive treatments; in curative assays, indices were 44% and 54%, respectively. This indicates that it is a promising bioactive compound to control cotton ramulosis.

Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20WW (Erg20F96W-N127W), co-expression of the reverse fusion of Erg20ww/t3CrGES and another copy of Erg20WW promoted the geraniol titer to 523.96mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20WW and the free Erg20WW. Eventually, a highest reported titer of 1.68g/L geraniol in eukaryote cells was achieved in 2.0L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.

Directed evolution and expression tuning of geraniol synthase for efficient geraniol production in Escherichia coli.

To achieve an efficient production of geraniol and its derivatives in Escherichia coli, we aimed to improve the activity of geraniol synthase (GES) through a single round of mutagenesis and screening for higher substrate consumption. We isolated GES variants that outperform their parent in geraniol production. The analysis of GES variants indicated that the expression level of GES was the bottleneck for geraniol synthesis. Over-expression of the mutant GESM53 with a 5'-untranslated sequence designed for high translational efficiency, along with the additional expression of mevalonate pathway enzymes, isopentenyl pyrophosphate isomerase, and geranyl pyrophosphate synthase, yielded 300 mg/L/12 h geraniol and its derivatives (>1000 mg/L/42 h in total) in a shaking flask.

Elevated CO2 improves growth, modifies anatomy, and modulates essential oil qualitative production and gene expression in Lippia alba (Verbenaceae)

Carbon dioxide (CO2) concentrations have grown in recent decades and will continue to increase during this century, affecting plant physiology and development. Aiming to evaluate the effect of CO2 elevation on growth, anatomy, essential oil qualitative production and expression of genes related to biosynthesis of these compounds, three chemotypes of Lippia alba (BGEN-01, BGEN-02 and BGEN-42) were cultivated in vitro. Firstly, we focused on the effects of gas exchange in the essential oil profile by comparing three CO2 exchange rates: 14, 21 and 25 µL L−1 s−1 CO2. Continuing, in addition to the previous 14 and 25 µL L−1 s−1 CO2 treatments, plants were placed into acrylic chambers with continuous forced air at 360 and 1000 μL L−1 of CO2; an additional control without allowing gas exchange was added inside the chambers, totaling five treatments with 6 replicates. After 45 days, essential oil profile, histochemical, stomatal density, growth evaluation analyses and transcript analysis were performed. The enrichment with CO2 enhanced plant dry and fresh weight, total chlorophylls and carotenoids in BGEN-01 and BGEN-02, and also increased stomatal density and lignin content for all chemotypes. The multivariate analysis showed that the essential oil profile varied, not only among the different chemotypes, but also within BGEN-01 and BGEN-02 treatments. The qualitative production was different in the treatments with forced air renovation and CO2 enrichment. Regarding the gene expression analyses, Farnesyl pyrophosphate synthase (LaFPPS) and Geranyl pyrophosphate synthase (LaGPPS) did not vary, except for the treatments with forced air ventilation (360 and 1000 µL L−1) in the BGEN-01, which had LaFPPS upregulated. Geraniol synthase (LaGES) was upregulated in all BGEN-02 treatments and for BGEN-01 treatments with 360 and 1000 µL L−1 CO2. Nerolidol/Linalool synthase (LaNES/LIS) was upregulated only in the BGEN-01, in the 360 and 1000 µL L−1 CO2 treatments. These findings provide a better understanding of how CO2 regulates secondary metabolites production, providing a basis to clarify the pathway regulation, further enabling the targeted production of essential oils with greater economic and industrial interest.

Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture.

BackgroundGeraniol is an acyclic monoterpene alcohol, which exhibits good prospect as a gasoline alternative. Geraniol is naturally encountered in plants at low concentrations and an attractive target for microbial engineering. Geraniol has been heterologously produced in Escherichia coli, but the low titer hinders its industrial applications. Moreover, bioconversion of geraniol by E. coli remains largely unknown.ResultsRecombinant overexpression of Ocimum basilicum geraniol synthase, Abies grandis geranyl diphosphate synthase, and a heterotic mevalonate pathway in E. coli BL21 (DE3) enabled the production of up to 68.6 ± 3 mg/L geraniol in shake flasks. Initial fed-batch fermentation only increased geraniol production to 78.8 mg/L. To further improve the production yield, the fermentation conditions were optimized. Firstly, 81.4 % of volatile geraniol was lost during the first 5 h of fermentation in a solvent-free system. Hence, isopropyl myristate was added to the culture medium to form an aqueous-organic two-phase culture system, which effectively prevented volatilization of geraniol. Secondly, most of geraniol was eventually biotransformed into geranyl acetate by E. coli, thus decreasing geraniol production. For the first time, we revealed the role of acetylesterase (Aes, EC 3.1.1.6) from E. coli in hydrolyzing geranyl acetate to geraniol, and production of geraniol was successfully increased to 2.0 g/L under controlled fermentation conditions.ConclusionsAn efficient geraniol production platform was established by overexpressing several key pathway proteins in engineered E. coli strain combined with a controlled fermentation system. About 2.0 g/L geraniol was obtained using our controllable aqueous-organic two-phase fermentation system, which is the highest yield to date. In addition, the interconversion between geraniol and geranyl acetate by E. coli was first elucidated. This study provided a new and promising strategy for geraniol biosynthesis, which laid a basis for large-scale industrial application.

Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.