Engineering Terpene Production Pathways in Methylobacterium extorquens AM1.

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.

PatWRKY71 transcription factor regulates patchoulol biosynthesis and plant defense response.

Patchoulol, a valuable compound belonging to the sesquiterpenoid family, is the primary component of patchouli oil produced by Pogostemon cablin (P. cablin). It has a variety of pharmacological and biological activities and is widely used in the medical and cosmetic industries. However, despite its significance, there is a lack of research on the transcriptional modulation of patchoulol biosynthesis.Salicylic acid (SA), is a vital plant hormone that serves as a critical signal molecule and plays an essential role in plant growth and defense. However, to date, no studies have explored the modulation of patchoulol biosynthesis by SA. In our study, we discovered that the application of SA can enhance the production of patchoulol. Utilizing transcriptome analysis of SA-treated P. cablin, we identified a crucial downstream transcription factor, PatWRKY71. The transcription level of PatWRKY71 was significantly increased with the use of SA. Furthermore, our research has revealed that PatWRKY71 was capable of binding to the promoter of PatPTS, ultimately leading to an increase in its expression. When PatWRKY71 was silenced by a virus, the expression of both PatWRKY71 and PatPTS was reduced, resulting in the down-regulation of patchoulol production. Through our studies, we discovered that heterologous expression of PatWRKY71 leads to an increase in the sensitivity of Arabidopsis to salt and Cd, as well as an outbreak of reactive oxygen species (ROS). Additionally, we uncovered the regulatory role of PatWRKY71 in both patchoulol biosynthesis and plant defense response. This discovery provided a theoretical basis for the improvement of the content of patchoulol and the resistance of P. cablin through genetic engineering.

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.

Metabolic flux enhancement from the translational fusion of terpene synthases is linked to terpene synthase accumulation.

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.

High-Level Production of Patchoulol in Yarrowia lipolytica via Systematic Engineering Strategies.

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 Cu2+-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.

Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii.

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.

Biomass generation and heterologous isoprenoid milking from engineered microalgae grown in anaerobic membrane bioreactor effluent.

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 CO2 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 CO2 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.

Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae / 中国中药杂志

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.

PatDREB Transcription Factor Activates Patchoulol Synthase Gene Promoter and Positively Regulates Jasmonate-Induced Patchoulol Biosynthesis.

The production of patchoulol in the patchouli (Pogostemon cablin) plant determines its application value, as it is the principal active sesquiterpene of essential oil extracted from this plant. Here, the promoter of patchoulol synthase gene (PatPTSpro) was isolated and found to be methyl jasmonate (MeJA)-induced. A nucleus-localized AP2/ERF transcription factor PatDREB was identified as a transcription activator binding to PatPTSpro, regulating patchoulol biosynthesis through modulating the gene expression. PatDREB also interacts with jasmonate ZIM-domain 4 (JAZ4). Furthermore, PatDREB could physically interact with the MYB-related transcription factor PatSWC4 and synergistically facilitate patchoulol biosynthesis. However, the transcriptional activation activity of the PatDREB-PatSWC4 complex could be inhibited by PatJAZ4, and JA could reverse this interference. Overall, we demonstrated the positive roles of PatDREB and the PatDREB-PatSWC4 complex in regulating patchoulol production, which advance our understanding of the regulatory network of patchoulol biosynthesis.

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.

Production of sesquiterpene patchoulol in mitochondrion-engineered Saccharomyces cerevisiae.

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.

Enhancement of Patchoulol Production in Escherichia coli via Multiple Engineering Strategies.

As a natural sesquiterpene compound with numerous biological activities, patchoulol has extensive applications in the cosmetic industry and potential usage in pharmaceuticals. Although several patchoulol-producing microbial strains have been constructed, the low productivity still hampers large-scale fermentation. Escherichia coli possesses the ease of genetic manipulation and simple nutritional requirements and does not comprise competing pathways for the farnesyl diphosphate (FPP) precursor, showing its potential for patchoulol biosynthesis. Here, combinatorial strategies were applied to produce patchoulol in E. coli. The initial strain was constructed, and it produced 14 mg/L patchoulol after fermentation optimization. Patchoulol synthase (PTS) was engineered by semirational design, resulting in improved substrate binding affinity and a patchoulol titer of 40.3 mg/L; the patchoulol titer reached 66.2 mg/L after fusing of PTS with FPP synthase. To further improve the patchoulol production, the genome of an efficient chassis strain was engineered by deleting the competitive routes for acetate, lactate, ethanol, and succinate synthesis and cumulatively enhancing the expression of efflux transporters, which improved patchoulol production to 338.6 mg/L. When tested in a bioreactor, the patchoulol titer and productivity were further improved to 970.1 mg/L and 199 mg/L/d, respectively, and were among the highest levels reported using mineral salt medium.

High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae.

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

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.

Construction of yeast producing patchoulol by global metabolic engineering strategy.

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.

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase.

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (-)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.

High-Level Patchoulol Biosynthesis in Artemisia annua L.

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.

Engineered Rhodobacter capsulatus as a Phototrophic Platform Organism for the Synthesis of Plant Sesquiterpenoids.

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 P nif 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.