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Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Patchoulol is a natural sesquiterpene alcohol with extensive applications in cosmetics and pharmaceuticals. In this study, we first constructed the synthesis pathway of patchoulol in Saccharomyces cerevisiae by expressing the patchoulol synthase PTS gene using the strong promoter GAL1. Afterward, the metabolic flux of the precursor was enhanced by strengthening the mevalonate pathway and balancing the precursor competition pathway, resulting in a 32.74-fold increase in patchoulol production. Subsequently, the supply of acetyl-CoA in yeast was increased by modifying transcriptional regulators and modulating the acetyl-CoA pathway, and the titer of patchoulol reached 155.94 mg/L. Finally, optimization of the fermentation conditions resulted in a titer of 195.96 mg/L in the shake flasks. Further, batch-fed fermentation in a 5 L bioreactor yielded 1.95 g/L. This work accelerated the development of a microbial cell factory for the production of patchoulol. [ABSTRACT FROM AUTHOR]

Patchoulol is a natural sesquiterpene alcohol with extensive applications in cosmetics and pharmaceuticals. In this study, we first constructed the synthesis pathway of patchoulol in Saccharomyces cerevisiae by expressing the patchoulol synthase PTS gene using the strong promoter GAL1. Afterward, the metabolic flux of the precursor was enhanced by strengthening the mevalonate pathway and balancing the precursor competition pathway, resulting in a 32.74-fold increase in patchoulol production. Subsequently, the supply of acetyl-CoA in yeast was increased by modifying transcriptional regulators and modulating the acetyl-CoA pathway, and the titer of patchoulol reached 155.94 mg/L. Finally, optimization of the fermentation conditions resulted in a titer of 195.96 mg/L in the shake flasks. Further, batch-fed fermentation in a 5 L bioreactor yielded 1.95 g/L. This work accelerated the development of a microbial cell factory for the production of patchoulol.

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels – resulting from improved expression and/or improved protein stability – is a major driver of catalytic enhancement from enzyme fusion. • Fusion of farnesyl diphosphate synthase (FPPS) to nerolidol synthase increased nerolidol production by >110-fold. • Fusion of nerolidol synthase to metabolically inactive proteins also increased titres. • Fusion of FPPS to other terpene synthases produced smaller increases in titre. • Increases in titre coincided with elevated levels of terpene synthase. [ABSTRACT FROM AUTHOR]

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Patchouli is a prized tropical medicinal herb with broad-spectrum therapeutic importance. The present research work describes development of an efficient callus-mediated plant regeneration protocol along with associated germplasm portability system (via alginate-encapsulation). Using 1.5 mg/l α-naphthalene acetic acid (NAA) and 1.0 mg/l 2, 4-dichlorophenoxy acetic acid (2, 4-D), highly proliferative friable calli were produced that subsequently underwent organogenesis in combinatorial cytokinin treatment to yield multiple shoot clusters. The highest frequency of shoot formation was achieved using 1.5 mg/l NAA with 1.5 mg/l 6-benzylaminopurine (BAP) in Murashige and Skoog (MS) medium. In vitro-derived shoot tips were encapsulated with 3% sodium alginate and 100 mM CaCl2 solution. The encapsulated beads were germinated in MS media with various concentrations of polyamines, where the highest regeneration frequency was observed with 1.5 mg/l spermidine. The regenerated shoots were rooted in basal MS medium and were successfully acclimatized with 96% survival rate. Genetic homogeneity amongst the regenerated plantlets was validated using Start Codon Targeted polymorphism (SCoT) and CAAT box-derived polymorphism (CBDP) ascertaining a high degree of clonal fidelity. The essential oil (EO) profiling of the donor plant and the in vitro-derived plantlets revealed identical composition. Furthermore, the antibacterial activities of various tissue extracts and extracted EOs were evaluated against the opportunistic pathogens viz. Klebsiella pneumoniae (MTCC 109), Salmonella typhii (MTCC 733), Micrococcus luteus (MTCC 2470) and Staphylococcus aureus (MTCC 96). The minimum inhibitory concentration (MIC) ranged from 0.31 to 5.0 mg/ml and 2.5 to 5.0 mg/ml against Gram-positive and Gram-negative bacteria, respectively. Eventually, the present research provides a holistic insight into the rapid regeneration of quality planting material as well as pharmacological bioprospection of patchouli along with the scope of further qualitative improvement via genetic transformation. [ABSTRACT FROM AUTHOR]

Mitochondria are a potential sub-organelle for the synthesis of terpenoids due to its abundant acetyl-CoA, cofactors, and compact compartment for targeting metabolic pathways and enzymes. Hence, conducting systematic investigations and harnessing the subcellular compartment of mitochondria may offer a viable avenue for enhancing terpenoids biosynthesis in yeast and other eukaryotic cells. Previous endeavors to boost terpenoids production in Komagataella phaffii mainly focused on diverse metabolic engineering strategies in the cytoplasm. Numerous studies have demonstrated that reconstructing specific metabolic pathways within mitochondria can lead to higher production yields than engineering the same pathways in the cytoplasm. In our study, we initially identified 6 well-performed mitochondrial targeting sequences (MTSs) through colocalization observation of EGFP and a mitochondrial red fluorescent probe via fluorescence microscopy. These MTSs were selected for the assembly of complete patchoulol biosynthesis pathways in the mitochondria, which resulted in a 1.7-fold increase in patchoulol production compared to the original strain. Furthermore, dual regulation of the cytoplasm and mitochondria was implemented in the P6H4＋PM2 strain to further enhance patchoulol synthesis to 109 mg/L, representing a 1.0-fold and 10.7-fold greater yield than that of the strains engineered solely in the cytoplasm or mitochondria, respectively. This study offers valuable guidance for effectively utilizing mitochondria to achieve higher patchoulol and other sesquiterpenes through dual cytoplasmic-mitochondrial engineering in K. phaffii. [Display omitted] • Endogenous mitochondrial targeting sequences were identified in K. phaffii. • This study represented the first attempt to synthesize patchoulol through mitochondrial compartmentalization in K. phaffii. • Dual regulation of cytoplasmic-mitochondrial engineering shows potential for enhancing patchoulol biosynthesis in K. phaffii. [ABSTRACT FROM AUTHOR]

Essential oils are gaining popularity for their use in treating depression, including that extracted from patchouli leaves and stems (Pogostemon cablin). Herein, we used patchouli oil (PO) containing a high amount of patchouli alcohol derived from P. cablin var. Tapak Tuan. The aim of this study was to investigate the antidepressant potential of PO, with a variety of patchouli alcohol concentrations obtained from a separation process using vacuum distillation with different temperature ranges. The initial patchouli oil (iPO) was traditionally distilled by a local farmer and further distilled using a rotary evaporator at temperature ranges of 115–160 °C (POF-1); 120–160 °C (POF-2), and 125–160 °C (POF-3), resulting in products with different patchouli alcohol concentrations. POF-3, with the highest patchouli alcohol content of 60.66% (based on gas chromatography-mass spectrometry), was used for cooling crystallization, resulting in 100% patchouli alcohol crystal (pPA). A tail suspension test (TST) was performed on a rat model to screen the antidepressant potential of iPO and its derivatives. The TST results revealed that POF-3 had the best antidepressant-like effect and was second only to the fluoxetine-based antidepressant, Kalxetin®, where both groups had significant reductions of immobility time post-treatment (p < 0.0001). Other than patchouli alcohol, POF-3 also contained ledol and trans-geraniol, which have been reported for their antidepressant-related activities. Brain dopamine levels increased significantly in the group treated with POF-3 (p < 0.05 as compared with the control group), suggesting its primary anti-depressant mechanism. These findings suggest the potential of vacuum-distilled patchouli oil in reducing depression via dopamine elevation. [ABSTRACT FROM AUTHOR]

Pogostemon cablin (Blanco) Benth. is an aromatic species popularly known as patchouli. The essential oil rich in patchoulol extracted from leaves is used by the pharmaceutical industries. The objective was to investigate the effects of shade nets and shading intensities on P. cablin growth, anatomy, chemical composition and essential oil content. The experiment was conducted with two types of shade nets (black and Aluminet), three light intensities (30, 50, and 70% shading) and full sun. The different light intensities influenced the growth and the essential oil yield, and chemical composition. Patchouli plants grown in an environment with shading showed higher values for the leaf, stem, root ant total dry weights compared to full sun. At 50% of shading, Aluminet provided gain in stem and total dry weights. Aluminet and black net at 50 and 70% of shading enhance essential oil yield. Patchoulol and pogostol contents were higher under full sun, and, black net and Aluminet at 50% shading. The environment for patchouli cultivation can be improved by use shade nets, especially with Aluminet at 50% of shading. [ABSTRACT FROM AUTHOR]

Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work. [ABSTRACT FROM AUTHOR]

Aromatic plants are known for the production of phytochemicals like essential oils which have several biological activities and have convincing fragrances. A frequent growth in the worldwide market of essential oil has been observed as it holds various beneficial therapeutic properties, which make them useful in food, fragrances, and medicinal sectors. The source of the patchouli oil isPogostemon cablin Benth, belonging to Lamiaceae family. Due to its lifelong and preserving properties, the oil has huge importance in aromatherapy and perfumery. Patchouli oil is the blend of various sesquiterpenes in which patchoulol (tricyclic sesquiterpenes) is the major constituent and the primary reason for the traditional aroma of the oil. This review article gives an overview on the importance of patchouli oil, major components of the oil, techniques employed to extract oil, its biological activities, the methods to isolate patchoulol from oil, and utilization of waste generated after the extraction. [ABSTRACT FROM AUTHOR]

Patchouli (Pogostemon cablin Benth.) is an industrially valued aromatic and important medicinal plant currently having a huge demand for its essential oil. It is widely used in flavor and fragrance industries as well as in pharmaceuticals. The essential oil is mainly isolated from the dried leaf of patchouli by steam distillation. Patchoulol and α-patchoulene are the major constituents that regulate and control patchouli essential oil quality. The major compounds of this essential oil are patchouli alcohol, α-guaiene, α-bulnesene, β-caryophyllene, α-patchoulene, γ-curcumene and some of the minor compounds are pogostone, limonene, cedrene, viridiflorol, γ-himachalene, etc. The patchouli essential oil is highly valued in perfumery and aromatherapy and is known to have various pharmacological activities such as anti-inflammatory, antiseptic, astringent, diuretic sedative, antimutagenic, antiviral, etc. Realizing the importance and industrial value of the crop, farmers are showing interest in the plantation of patchouli at a commercial scale. In this review, a broad scientific literature were looked through which incorporates Sci-hub, Pub Med, Google Scholar, Pub Chem, NIST chemistry web book and NCBI. The purpose of the review is to summarize the information's available includes Pogostemon cablin essential oil (EO) extraction and compositions, qualitative and quantitative analysis of the EOs, pharmacological importance, high essential oil yielding variety, adulteration, authentication, patchouli cultivation practices, and industrial demand of patchouli essential oil. An attempt has been made to enlighten the work of various researchers on patchouli carried out to date. [ABSTRACT FROM AUTHOR]

ABSTRACT: Pogostemon cablin (Blanco) Benth. is an aromatic species popularly known as patchouli. The essential oil rich in patchoulol extracted from leaves is used by the pharmaceutical industries. The objective was to investigate the effects of shade nets and shading intensities on P. cablin growth, anatomy, chemical composition and essential oil content. The experiment was conducted with two types of shade nets (black and Aluminet), three light intensities (30, 50, and 70% shading) and full sun. The different light intensities influenced the growth and the essential oil yield, and chemical composition. Patchouli plants grown in an environment with shading showed higher values for the leaf, stem, root ant total dry weights compared to full sun. At 50% of shading, Aluminet provided gain in stem and total dry weights. Aluminet and black net at 50 and 70% of shading enhance essential oil yield. Patchoulol and pogostol contents were higher under full sun, and, black net and Aluminet at 50% shading. The environment for patchouli cultivation can be improved by use shade nets, especially with Aluminet at 50% of shading.

Abstract Background Many commercially important drug and flavor compounds are secondary metabolites of terpenoid origin. Pogostemon cablin, a commercially important industrial and medicinal crop, accumulates abundant patchouli oil comprised of more than 24 unique sesquiterpene compounds, with the most abundant being patchouli alcohol. Results In this study, we analyzed the P. cablin transcriptome library, obtaining 74 terpenoid biosynthesis-related genes, and identified their expression patterns in leaves, stems, and flowers. These genes are members of 15 different families, and we detected all the enzymes involved in the sesquiterpenes pathway that are responsible for patchoulol biosynthesis. Sequence structure, homology, conserved domain properties, and phylogeny of certain identified genes were systematically investigated. Color complementation assay was used to verify the functional activity of the MEP pathway proteins. Exogenous hormone treatment revealed that patchoulol synthesis is induced by methyl jasmonate (MeJA). Quantitative reverse-transcription PCR analysis indicated that the MVA pathway genes (acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, mevalonate diphosphate decarboxylase, and farnesyl diphosphate synthase) participate in patchoulol biosynthesis and are mediated by MeJA. Conclusions Taken together, this is the first report of integrated analysis of P. cablin MVA and MEP pathway related genes, providing a better understanding of terpenoid and/or patchoulol biosynthesis in P. cablin, and the basis for improving patchoulol production through genetic engineering.

Abstract Background Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. Results In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. ‘Zhanxiang’, and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. Conclusions The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. ‘Zhanxiang’, these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.

Terpenes constitute the largest class of secondary metabolites in plants. Some terpenes are essential for plant growth and development, membrane components, and photosynthesis. Terpenes are also economically useful for industry, agriculture, and pharmaceuticals. However, there is very low content of most terpenes in microbes and plants. Chemical or microbial synthesis of terpenes are often costly. Plants have the elaborate and economic biosynthetic way of producing high-value terpenes through photosynthesis. Here we engineered the heterogenous sesquiterpenoid patchoulol production in A. annua. When using a strong promoter such as 35S to over express the avian farnesyl diphosphate synthase gene and patchoulol synthase gene, the highest content of patchoulol was 52.58 μg/g DW in transgenic plants. When altering the subcellular location of the introduced sesquiterpene synthetase via a signal peptide, the accumulation of patchoulol was observably increased to 273 μg/g DW. This case demonstrates that A. annua plant with glandular trichomes is a useful platform for synthetic biology studies.

Essential oils are gaining popularity for their use in treating depression, including that extracted from patchouli leaves and stems (Pogostemon cablin). Herein, we used patchouli oil (PO) containing a high amount of patchouli alcohol derived from P. cablin var. Tapak Tuan. The aim of this study was to investigate the antidepressant potential of PO, with a variety of patchouli alcohol concentrations obtained from a separation process using vacuum distillation with different temperature ranges. The initial patchouli oil (iPO) was traditionally distilled by a local farmer and further distilled using a rotary evaporator at temperature ranges of 115–160 °C (POF-1); 120–160 °C (POF-2), and 125–160 °C (POF-3), resulting in products with different patchouli alcohol concentrations. POF-3, with the highest patchouli alcohol content of 60.66% (based on gas chromatography-mass spectrometry), was used for cooling crystallization, resulting in 100% patchouli alcohol crystal (pPA). A tail suspension test (TST) was performed on a rat model to screen the antidepressant potential of iPO and its derivatives. The TST results revealed that POF-3 had the best antidepressant-like effect and was second only to the fluoxetine-based antidepressant, Kalxetin®, where both groups had significant reductions of immobility time post-treatment (p < 0.0001). Other than patchouli alcohol, POF-3 also contained ledol and trans-geraniol, which have been reported for their antidepressant-related activities. Brain dopamine levels increased significantly in the group treated with POF-3 (p < 0.05 as compared with the control group), suggesting its primary anti-depressant mechanism. These findings suggest the potential of vacuum-distilled patchouli oil in reducing depression via dopamine elevation.

Patchouli [Pogostemon cablin (Blanco) Benth.] is a plant of the family Lamiaceae, widely used as an essential oil in the cosmetics and perfumery industry. This study aimed to evaluate the influence of storage time and temperature of dry leaves on the patchouli essential oil content and chemical composition. The experiment was performed in a completely randomized design in a 6x2x2 factorial scheme, testing storage time (0, 1, 2, 4, 8, and 16 weeks) and temperature (28°C and 33°C) of dry leaves of two patchouli genotypes (POG-015 and POG-021). The variables essential oil content and chemical composition, and the identification of fungus during storage were evaluated. Results showed that the storage significantly influenced the essential oil content. Patchoulol was identified as the major compound in both genotypes, ranging from 55.05% to 68.77% (POG-15) and from 52.83% to 64.06% (POG-021). Based on the results of patchoulol, dry leaves of both genotypes (POG-015 and POG-021) can be stored for up to eight weeks at 28- 33°C without altering the essential oil quality.

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Patchoulol is a sesquiterpene alcohol found in the leaves of the patchouli plant that can be extracted by steam distillation. Notably, patchoulol is an essential natural product frequently used in the chemical industry. However, patchouli produces an insignificant amount of patchoulol, not to mention steam distillation, and requires a lot of energy and time. Recombinant microorganisms that can be cultured in mild conditions and can produce patchoulol from renewable biomass resources may be a promising alternative. We previously developed the global metabolic engineering strategy (GMES), which produces a comprehensive metabolic modification in yeast, using the cocktail δ‐integration method. In this study, we aimed to produce patchoulol by modifying engineered yeast. The expression of nine genes involved in patchoulol synthesis was modulated using GMES. Regarding patchoulol production, the resultant strain, YPH499/PAT167/MVA442, showed a concentration of 42.1 mg/L, a production rate of 8.42 mg/L/d, and a yield of 2.05 mg/g‐glucose, respectably. These concentration values, production rate, and yield obtained through batch‐fermentation in this study were high level when compared to previously reported recombinant microorganism studies. GMES could be used as a potential strategy for producing secondary metabolites from plants in recombinant Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

[Display omitted] • Engineering patchoulol synthase with 2.02-fold higher patchoulol titer. • Engineering carbon catabolite repression for sesquiterpene production. • Exploiting dynamic glycolysis regulation device for overflow metabolism. • Highest patchoulol production in Escherichia coli. • First report for microbial production of τ-cadinol with high titer. Glucose metabolism suppresses the microbial synthesis of sesquiterpenes with a syndrome of " too much of a good thing can be bad ". Here, patchoulol production in Escherichia coli was increased 2.02 times by engineering patchoulol synthase to obtain an initial strain. Knocking out the synthetic pathway for cyclic adenosine monophosphate relieved glucose repression and improved patchoulol titer and yield by 27.7 % and 43.1 %, respectively. A glycolysis regulation device mediated by pyruvate sensing was constructed which effectively alleviated overflow metabolism in a high-glucose environment with 10.2 % greater patchoulol titer in strain 070QA. Without fine-tuning the glucose-feeding rate, patchoulol titer further increased to 1675.1 mg/L in a 5-L bioreactor experiment, which was the highest level reported in E. coli. Using strain 070QA as a chassis, the τ-cadinol titer reached 15.2 g/L, representing the first report for microbial production of τ-cadinol. These findings will aid in the industrial production of sesquiterpene. [ABSTRACT FROM AUTHOR]

Sesquiterpenoids are a large class of natural compounds offering manifold properties valuable for food, cosmetics, agriculture, and pharma industry. Production in microorganisms is a sustainable approach to provide sesquiterpenoids for research and industrial use independent of their natural sources. This requires the functional transfer of the respective biocatalytic pathways in an adequate host microorganism offering a sufficient supply of precursors that is ideally adjusted to the individual demand of the recombinant biosynthesis route. The phototrophic purple bacterium Rhodobacter capsulatus offers unique physiological properties that are favorable for biosynthesis of hydrophobic terpenes. Under phototrophic conditions, it develops a large intracytoplasmic membrane suitable for hosting membrane-bound enzymes and metabolites of respective biosynthetic pathways. In addition, Rhodobacter harbors an intrinsic carotenoid biosynthesis that can be engineered toward the production of foreign terpenes. Here, we evaluate R. capsulatus as host for the production of plant sesquiterpenoids under phototrophic conditions using patchoulol and valencene as a proof of concept. The heterologous expression of patchoulol synthase PcPS from Pogostemon cablin as well as the valencene synthases CsVS from Citrus sinensis and CnVS from Callitropsis nootkatensis led to the production of the respective sesquiterpenoids in R. capsulatus. To analyze, if gradually adjustable formation of the key precursor farnesylpyrophosphate (FPP) is beneficial for sesquiterpene synthesis under phototrophic conditions, the intrinsic 1-deoxy-D-xylulose 5-phosphate (DXP) pathway genes as well as the heterologous mevalonate pathway genes were modularly expressed in various combinations. To this end, different plasmids and chromosomally integrated expression tools were developed harboring the strong and tightly controlled Pnif promoter for heterologous gene expression. Notably, comparative studies identified a distinct combination of precursor biosynthetic genes as best-performing setup for each of the tested sesquiterpene synthases. In summary, we could demonstrate that R. capsulatus is a promising alternative platform organism that is suited for sustainable sesquiterpenoid formation under phototrophic cultivation conditions. A modular engineering of R. capsulatus strains via tailored co-expression of FPP biosynthetic genes further allowed adaptation of sesquiterpene precursor formation to its catalytic conversion by different plant terpene synthases.

RESUMOPatchouli é uma espécie aromática pertencente à família Lamiaceae que possui óleo essencial com alto valor comercial devido à aplicação nas indústrias de higiene, cosméticos e perfumarias. Considerando as características voláteis e estruturas de armazenamento em diferentes órgãos das plantas aromáticas, a determinação de métodos de secagem é de extrema importância e pode afetar tanto o rendimento como a composição do óleo essencial. Este trabalho teve como objetivo principal avaliar o teor e a composição do óleo essencial de patchouli após secagem por diferentes períodos em estufa e a temperatura ambiente. O experimento foi realizado durante o mês de fevereiro de 2011 em delineamento experimental inteiramente casualizado em esquema fatorial 2X6, correspondente a dois métodos de secagem (estufa 65ºC e temperatura ambiente) e seis tempos para o início da extração do óleo essencial após a (0, 2, 4,6,8 e 10 dias após a colheita),com três repetições. O óleo essencial foi extraído por meio hidrodestilação em aparelho graduado de Clevenger e os constituintes do óleo essencial foram analisados por cromatografia em fase gasosa acoplada à espectrometria de massas. Houve interação significativa entre os métodos e tempos de secagem tanto para o teor como para a composição do óleo essencial. A secagem das plantas por dois dias em estufa a 65ºC aumentou o teor de óleo essencial, após este período houve redução significativa. Para as folhas secas em temperatura ambiente, o teor de óleo essencial foi superior aos quatro e aos seis dias após a colheita. Comparando-se os métodos, a secagem em estufa mostrou-se mais eficiente, sendo o teor significativamente superior após dois dias de secagem. Após este período, no entanto, houve drástica redução do teor de óleo essencial, sendo inferior ao teor observado em todos os períodos nas folhas secas em temperatura ambiente. Os diferentes métodos e tempos de secagem alteraram também a composição do óleo essencial. Embora a secagem em estufa a 65º por dois dias não tenha resultado em redução dos teores dos constituintes majoritários patchoulol e pogostol, maiores teores foram observados após seis ou oito dias de secagem. Quando a secagem foi realizada a temperatura ambiente, não houve alteração dos teores de ambos os constituintes.

ABSTRACT The purpose of this investigation was to determine the influence of colored shade nets on the growth, anatomy and essential oil content, yield and chemical composition of Pogostemon cablin. The plants were cultivated under full sunlight, black, blue and red nets. The harvesting was performed 5 months after planting and it was followed by the analysis of plant growth parameters, leaf anatomy, essential oil content, yield and chemical composition. The plants grown under red net have produced more leaf, shoot, total dry weight and leaf area. Plants cultivated under colored nets showed differences in morphological features. Plants maintained under red net had a higher leaf blade thickness and polar and equatorial diameter of the stomata ratio. Additionally, higher yield of essential oil in the leaves was observed under red and blue colored shade net. The essential oil of the plants grown under red net showed the highest relative percentage of patchoulol (66.84%). Therefore, it is possible using colored shade nets to manipulate P. cablin growth, as well as its essential oil production with several chemical compositions. The analyses of principal components allowed observing that pogostol has negative correlation with α-guaiene and α-bulnesene. There was difference in total dry weight and patchoulol content when the patchouli is cultured under the red colored shade nets.

The leaves of Pogostemon cablin (Lamiaceae) are the source of patchouli essential oil, which is - with an annual production of about 1300 tonnes - an important and unique commodity in the fragrance industry. All the literature pertaining to patchouli was critically reviewed with an emphasis on the qualitative and quantitative chemical analysis of the oil but also harvesting, fermentation, drying, distillation, used analytical techniques, sensory aspects including molecules responsible for the odour, adulteration and toxicological aspects, i.e., skin sensitisation, are discussed. In total 72 constituents have been convincingly identified in the oil and another 58 tentatively. The main constituent is the sesquiterpene patchoulol. For this review over 600 papers were consulted and in the supplementary information all patchouli-related references not relevant enough to be cited in the paper itself are listed. [ABSTRACT FROM AUTHOR]

The title compound, C15H26O, commonly known as Patchouli alcohol or Patchoulol, is a tricyclo[5.3.1.03,8]undecane. It crystallized in the enantiomer-defining hexagonal space group P63. However, the absolute structure could not be determined [absolute structure parameter = 0.4 (10)]. In the crystal, three molecules are linked by O—H...O hydrogen bonds, forming a trimer with an R33(6) ring motif. The crystal structure of patchouli alcohol determined by the crystalline inclusion method, using 1,1,6,6-tetraphenylhexa-2,4-diyne-1,6-diol as host, has been reported [Tong et al., (2013). Nat. Prod. Res. 27, 32–36].

The essential oil from the leaf of Pogostemon cablin, grown in Indonesia was extracted by microwave-assisted hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS). Nineteen compounds were identified representing approximately 97.97% of the oil. The main compound of the oil was the oxygenated terpene patchoulol (26.32%). Other major compounds of the essential oil were δ-guaiene (14.69%), α-guaiene (12.18%), α-Gurjunene (11.13%), seychellene (8.42%), viridiflorol (5.93%), β-caryophyllene (4.63%) and β-patchoulene (2.87%). Four new compounds were found in this oil: 2(1H)-naphthalenone, octahydro-1-methyl-1-(2-propenyl)-, (1a,4ab,8aa)- (2.64%); iso-.aplha.-cedren-15-al (0.35%); 7-oxabicyclo[4.1.0]heptane, 1,3,3-trimethyl-2-(3-methyl-1,3-butadienyl)-, [1a,2b(Z),6a]- (0.12%) and hexahydrothunbergol (0.11%). [ABSTRACT FROM AUTHOR]

O patchouli possui óleo essencial nas folhas com utilização principalmente na indústria de perfumaria. O objetivo foi avaliar o melhor tempo de extração de óleo essencial de folhas secas de patchouli. Os tratamentos foram 1, 2, 3, 4, 5, 6, 7 e 8 horas de extração, através do método de hidrodestilação, com aparelho graduado do tipo Clevenger e balões com capacidade de 2 L.O delineamento foi inteiramente casualizado com três repetições. O material destilado foi seco à sombra até atingir aproximadamente 20% de umidade. Para cada tratamento, foram utilizadas amostras de 50 g de massa seca foliar. Foram avaliados o rendimento e a composição do óleo essencial. Não houve diferença entre os diferentes tempos de extração no rendimento de óleo essencial, podendo a extração do óleo essencial de patchouli ser realizada com uma hora de extração. O tempo de extração aumenta as porcentagens relativas do beta-guaieno (0,81%), beta-patchouleno (1,26%), alfa-selineno (1,37%), cariofileno (2,44%), alfa-patchouleno (3,08%) e gama-patchouleno (4,82%). O teor de pogostol (5,11%) reduz com o aumento do tempo de extração. O patchoulol, alfa-guaieno, alfa-bulneseno e seicheleno não sofrem influencia do tempo de extração.

• Engineered Chlamydomonas can grow directly in AnMBR effluent. • Algal strain grew in modeled environmental conditions of the central Red Sea coast. • Algae cleaned N and P from AnMBR effluent and CO 2 from off-gas. • Engineered strain produced a heterologous sesquiterpene and biomass from effluent. • Bio-processes with heavily engineered algae in AnMBR effluent are possible. Wastewater (WW) treatment in anaerobic membrane bioreactors (AnMBR) is considered more sustainable than in aerobic reactors. However, outputs from AnMBR are a mixed methane and carbon dioxide gas stream as well as ammonium- (N) and phosphate- (P) containing waters. Using AnMBR outputs as inputs for photoautotrophic algal cultivation can strip the CO 2 while removing N and P from effluent which feed algal biomass generation. Recent advances in algal engineering have generated strains that produce high-value side products concomitant with biomass, although only shown in heavily domesticated, lab-adapted strains. Here, it was investigated whether engineered Chlamydomonas reinhardtii could be grown directly in AnMBR effluent with CO 2 concentrations found in AnMBR off-gas. The strain was found to proliferate over bacteria in the non-sterile effluent, consume N and P to levels that meet general discharge or reuse limits, and tolerate cultivation in modelled (extreme) outdoor environmental conditions prevalent along the central Red Sea coast. In addition to ∼2.4 g CDW L–1 biomass production in 96 h, a high-value heterologous sesquiterpene co-product could be obtained from 'milking' up to 837 µg L–1 culture in 96 h. This is the first demonstration of a combined bio-process that employs a heavily engineered algal strain to enhance the product generation potentials from AnMBR effluent treatment. This study shows it is possible to convert waste into value through use of engineered algae while also improving wastewater treatment economics through co-product generation. [Display omitted] [ABSTRACT FROM AUTHOR]

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu 2+ -repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii . The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242 µg g −1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03 mg L −1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source. [ABSTRACT FROM AUTHOR]

Biocatalysis of patchoulol (PA) was performed by the fungus Cunninghamella echinulata var. elegans . Eight metabolites ( 1–8 ) including four new compounds were obtained, and their structures were elucidated as (5 R ,8 S )-5,8 dihydroxypatchoulol ( 1 ), (5 R *,9 R *)-5,9 dihydroxypatchoulol ( 2 ), (6 S *, 9 S *)-6,9 dihydroxypatchoulol ( 3 ), and (4 R *)-4 hydroxypatchoulol ( 4 ) by spectroscopic analysis. The absolute configuration of 1 was determined by single crystal X-ray diffraction. [ABSTRACT FROM AUTHOR]

The moss Physcomitrella patens, has been genetically engineered to produce patchoulol and β-santalene, two valuable sesquiterpenoid ingredients in the fragrance industry. The highest yield of patchoulol achieved was 1.34 mg/g dry weight. This was achieved by non-targeted transformation of the patchoulol synthase and either a yeast or P. patens HMGR gene under the control of a 35S promoter. Santalene synthase targeted to the plastids yielded 0.039 mg/g dry weight of α/β santalene; cytosolic santalene synthase and 35S controlled HMGR afforded 0.022 mg/g dry weight. It has been observed that the final yield of the fragrance molecules is dependent on the expression of the synthase. This is the first report of heterologous production of sesquiterpenes in moss and it opens up a promising source for light-driven production of valuable fragrance ingredients.

Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS , and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.

The patchoulol synthase (PTS) is a multi-product sesquiterpene synthases which is the central enzyme for biosynthesis of patchouli essential oil in the patchouli plant. Sesquiterpene synthases catalyse the formation of various complex carbon backbones difficult to approach by organic synthesis. Here, we report the characterisation of a recombinant patchoulol synthase complementary DNA (cDNA) variant (PTS var. 1), exhibiting significant amino acid exchanges compared to the native PTS. The product spectrum using the natural substrate E,E-farnesyl diphosphate (FDP) as well as terpenoid products resulting from conversions employing alternative substrates was analysed by GC-MS. In respect to a potential use as a biocatalyst, important enzymatic parameters such as the optimal reaction conditions, kinetic behaviour and the product selectivity were studied as well. Adjusting the reaction conditions, an increased patchoulol ratio in the recombinant essential oil was achieved. Nevertheless, the ratio remained lower than in plant-derived patchouli oil. As alternative substrates, several prenyl diposphates were accepted and converted in numerous compounds by the PTS var. 1, revealing its great biocatalytic potential. [ABSTRACT FROM AUTHOR]