Metabolic engineering of glycolysis in Escherichia coli for efficient production of patchoulol and τ-cadinol.

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.

[Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae].

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.

Indirect organogenesis-mediated high frequency conversion of non-embryonic synthetic seeds, essential oil profiling and antibacterial activity in genetically stable plants of Patchouli.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03302-3.

Antidepressant-like Activity of Patchouli Oil var. Tapak Tuan (Pogostemon cablin Benth) via Elevated Dopamine Level: A Study Using Rat Model.

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.

Influence of storage temperatures and storage time of dry leaves on patchouli [Pogostemon cablin (Blanco) Benth. ] essential oil / Influência do tempo e temperaturas de armazenamento das folhas secas no óleo essencial de patchouli [Pogostemon cablin (Blanco) Benth. ]

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.

Patchouli [Pogostemon cablin (Blanco) Benth.] é uma planta pertencente à família Lamiaceae, e seu óleo essencial é utilizado nas indústrias de perfumes e cosméticos. O objetivo do trabalho foi avaliar a influência do tempo e temperaturas de armazenamento das folhas secas no teor e na composição química do óleo essencial de patchouli. O delineamento experimental foi o inteiramente casualizado em esquema fatorial 6x2x2, testando tempo de armazenamento (0, 1, 2, 4, 8 e 16 semanas), temperatura de armazenamento (28°C e 33°C) de folhas secas de dois genótipos (POG-015 e POG-021) de patchouli. As variáveis avaliadas foram o teor e a composição química do óleo essencial e a identificação dos fungos que se desenvolveram durante o armazenamento. Os resultados mostraram que o armazenamento influenciou significativamente o teor de óleo essencial. Dos compostos identificados, o patchoulol foi o composto majoritário nos dois genótipos, variando de 55,05% a 68,77% (POG-15) e 52,83% a 64,06% (POG-021). Baseado no patchoulol, folhas secas dos genótipos (POG-015 e POG-021) de patchouli podem ser armazenadas por um período de até oito semanas em temperatura de 28 e 33°C, sem alterar a qualidade do óleo essencial.

Evaluation of Contact Toxicity and Repellency of the Essential Oil of Pogostemon cablin Leaves and Its Constituents Against Blattella germanica (Blattodae: Blattelidae).

The aim of this research was to evaluate contact toxicity and repellency of the essential oil of Pogostemon cablin (Blanco) Bentham leaves against German cockroaches (Blattella germanica) (L.) and to isolate any active constituents. Essential oil of P. cablin leaves was obtained by hydrodistillation and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Twenty-three components were identified in the essential oil, and the main constituents were patchoulol (41.31%), pogostone (18.06%), α-bulnesene (6.56%), caryophyllene (5.96%), and seychellene (4.32%). Bioactivity-directed chromatographic separation of the essential oil led to the isolation of pogostone, patchoulol, and caryophyllene as active compounds. The essential oil of P. cablin leaves exhibited acute toxicity against male B. germanica adults with an LC50 value of 23.45 µg per adult. The constituent compound, pogostone (LC50 = 8.51 µg per adult) showed stronger acute toxicity than patchoulol (LC50 = 207.62 µg per adult) and caryophyllene (LC50 = 339.90 µg per adult) against the male German cockroaches. The essential oil of P. cablin leaves and the three isolated constituents exhibited strong repellent activity against German cockroaches at a concentration of 5 ppm. The results indicated that the essential oil of P. cablin leaves and its major constituents have good potential as a source for natural insecticides and repellents.

Teor e composição do óleo essencial de patchouli (Pogostemon cablin (Blanco) Benth. ) após diferentes tempos de secagem em estufa e temperatura ambiente / Yield and composition of essential oil of patchouli (Pogostemon cablin (Blanco) Benth. ) after different drying periods in oven and at room temperatur

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.

ABSTRACTPatchouli is an aromatic species belonging to the Lamiaceae family that has essential oil with high commercial value to the hygiene, cosmetic and perfume industries. Considering its volatile characteristics and storage structures in different organs of aromatic plants, the determination of drying methods is extremely important and can affect both the yield and the composition of the essential oil. This work aimed to evaluate the content and composition of the patchouli essential oil after drying it in different periodin an oven and at room temperature. The trial was carried out in February 2011 at an experimental design completely randomized in 2X6 factorial scheme, corresponding to two drying methods (oven at 65˚C and room temperature) and six periods of essential oil extraction after harvest (0, 2, 4, 6, 8, and 10 days after harvest), with three replications. The essential oil was extracted by hydrodistillation in a Clevenger apparatus and the essential oil components were analyzed by gas chromatography coupled to mass spectrometry. There was significant interaction among the methods and drying times for both the content as well as for the composition of essential oil. The drying of the plants for two days in an oven at 65˚C increased the essential oil content; after this period, there was a significant reduction. For the leaves that dried at room temperature, their contentof essential oil was superior at the fourth and sixth days after the harvest. When comparing the methods, the drying in the oven was more efficient, with the oil content being significantly higher after two days of drying. After this period, however, there was a drastic reduction in the essential oil content, lower than the one observed in all periods when the leaves dried at room temperature. The different methods and drying periods also altered the essential oil`s composition. Although drying in oven at 65˚ for two days did not result in reduction of the contents of patchoulol and pogostol, higher contents were observed after six or eight days of drying. When drying was carried out at room temperature, no change in relative content of both components was observed.

Desenvolvimento vegetativo, rendimento e composição do óleo essencial do patchouli submetido a diferentes doses de nitrogênio no plantio e manutenção / Vegetative growth, yield and essential oil of patchouli subjected to different levels of nitrogen fertilizer in the planting and maintenance

Neste trabalho avaliou-se o efeito da aplicação no plantio e na manutenção de diferentes doses de nitrogênio (30, 60, 90 e 120 Kg ha-1) no desenvolvimento do patchouli, bem como no rendimento e composição do óleo essencial. O experimento foi conduzido no município de Joinville-SC, de agosto de 2006 a maio de 2007, com delineamento em blocos ao acaso em parcelas subdivididas no tempo, com cinco repetições. Foram executados duas épocas de adubação para avaliação dos níveis de nitrogênio, após o plantio e após primeiro corte, na manutenção. Avaliouse o acúmulo de massa seca foliar e de ramo, número e comprimento de ramos, área foliar da planta e rendimento e composição do óleo essencial. A aplicação de 98 Kg ha-1 de nitrogênio na manutenção proporcionaram maiores ganhos de massa seca foliar. Maior rendimento de óleo essencial foi obtido no segundo corte, após a adubação de manutenção. A composição do óleo essencial, aparentemente, não sofreu influência dos diferentes níveis de nitrogênio aplicados em ambas as épocas (plantio e manutenção). As médias do constituinte majoritário patchoulol variaram entre 45,66% e 49,54%.

This work evaluated the effect of the application in the planting and maintenance of different levels of nitrogen fertilization (30, 60, 90 and 120 Kg ha-1) in the development of patchouli. The experiment was carried out at Joinville-SC from August 2006 the May 2007. The experimental design was completely randomized blocks subdivided in time, with five replications. Two time fertilization were carried out to evaluate the levels of nitrogen, after planting and after first cut, in maintenance. It was determined the leaf and branch dry mass, number and length of branches, leaf area and essential oil yield and composition. The application of 98 Kg ha-1 of nitrogen in maintenance provided greater gains of leaf dry mass. Largest essential oil yield was obtained in the second cut, after fertilization. The composition of the essential oil was not affected by different nitrogen levels in both the times (plantation and maintenance). The averages of the main constituent patchoulol varied from 45,66% to 49.54%.

Rendimento e composição do óleo essencial de patchouli (Pogostemon cablin) conforme o tempo de extração / Yield and composition of the patchouli (Pogostemon cablin) essential oil according to the extraction time

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.

Patchouli accumulatea an essential oil on its leaves, and it is mainly used by the perfume industry. The purpose of this work was to determine the distillation time for the extraction of essential oils from the leaves of patchouli. The treatments included 1, 2, 3, 4, 5, 6, 7 and 8 hours of extraction through the hydrodistillation method, using a Clevenger apparatus. The experimental design was completely randomized with three replications. The leaves were dried at room temperature until they reached 20% of humidity. The essential oil yield was measured from samples containing 50g of leaf dry mass. The essential oil production and composition were evaluated. No differences among the treatments were found on the essential oil yield, suggesting that the essential oil extraction can be carried out for 1 hour according to the experimental conditions. However, extending the extraction time, an increase in the relative percentage of beta-patchoulene (1.26%), cariofilene (2.44%), gamma-patchoulene (4.82%), alpha-patchoulene (3.08%), beta-guaiene (0.81%) and alpha-selinene (1.37%) was observed. The pogostol (5.11%) content was reduced with the increase of the extraction time. Patchoulol, alpha-guaieno, alpha-bulneseno and seicheleno contents were not affected by the evaluated extraction times.