Limonene Production Research Articles

Optimizing HMG-CoA Synthase Expression for Enhanced Limonene Production in Escherichia coli through Temporal Transcription Modulation Using Optogenetics.

Overexpression of a single enzyme in a multigene heterologous pathway may be out of balance with the other enzymes in the pathway, leading to accumulated toxic intermediates, imbalanced carbon flux, reduced productivity of the pathway, or an inhibited growth phenotype. Therefore, optimal, balanced, and synchronized expression levels of enzymes in a particular metabolic pathway is critical to maximize production of desired compounds while maintaining cell fitness in a growing culture. Furthermore, the optimal intracellular concentration of an enzyme is determined by the expression strength, specific timing/duration, and degradation rate of the enzyme. Here, we modulated the intracellular concentration of a key enzyme, namely HMG-CoA synthase (HMGS), in the heterologous mevalonate pathway by tuning its expression level and period of transcription to enhance limonene production in Escherichia coli. Facilitated by the tuned blue-light inducible BLADE/pBad system, we observed that limonene production was highest (160 mg/L) with an intermediate transcription level of HMGS from moderate light illumination (41 au, 150 s ON/150 s OFF) throughout the growth. Owing to the easy penetration and removal of blue-light illumination from the growing culture which is hard to obtain using conventional chemical-based induction, we further explored different induction patterns of HMGS under strong light illumination (2047 au, 300 s ON) for different durations along the growth phases. We identified a specific timing of HMGS expression in the log phase (3-9 h) that led to optimal limonene production (200 mg/L). This is further supported by a mathematical model that predicts several periods of blue-light illumination (3-9 h, 0-9 h, 3-12 h, 0-12 h) to achieve an optimal expression level of HMGS that maximizes limonene production and maintains cell fitness. Compared to moderate and prolonged transcription (41 au, 150 s ON/150 s OFF, 0-73 h), strong but time-limited transcription (2047 au, 300 s ON, 3-9 h) of HMGS could maintain its optimal intracellular concentration and further increased limonene production up to 92% (250 mg/L) in the longer incubation (up to 73 h) without impacting cell fitness. This work has provided new insight into the "right amount" and "just-in-time" expression of a critical metabolite enzyme in the upper module of the mevalonate pathway using optogenetics. This study would complement previous findings in modulating HMGS expression and potentially be applicable to heterologous production of other terpenoids in E. coli.

Systems biology approach for enhancing limonene yield by re-engineering Escherichia coli

Engineered microorganisms have emerged as viable alternatives for limonene production. However, issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition may reduce yields. To understand the underlying metabolism, we adopted a systems biology approach for an engineered limonene-producing Escherichia coli strain K-12 MG1655. Firstly, we generated time-series metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to track the native metabolic networks and the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included 13C-tracer studies, we performed in silico knockouts (KOs) of all enzymes to identify bottleneck(s) for optimal limonene yields. The simulations indicated that ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase) suppression, and HK (hexokinase) enhancement would increase limonene yields. Experimental confirmation was achieved, where ALDH-ADH and LDH KOs, and HK overexpression improved limonene yield by 8- to 11-fold. Our systems biology approach can guide microbial strain re-engineering for optimal target production.

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies

BackgroundLimonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.ResultsIn this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.ConclusionsIn this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides.

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

Turning Waste into Wealth: A Conceptual Design of Limonene Production Plant from Waste Rubber Tyre

Limonene production represents a compelling opportunity in the global market, given its competitive landscape dominated by a few key players who control nearly half of the industrial-grade limonene market. Due to the high polyisoprene content in vehicle tyres, along with the rising number of waste rubber tyres, the production of limonene by pyrolysis of tyres may represent a future trend to turn waste into wealth. Hence, a conceptual plant design was carried out to determine the feasibility of limonene production from waste rubber tyres in Malaysia. This feasibility study involved Aspen Plus simulation and illustrated the main processes using Process Flow Diagram (PFD), Piping and Instrumentation Diagram (P&ID), and comprehensive stream table integrating material and energy balances. The desired product, which is limonene, was produced by the gas-solid pyrolysis within a conical spouted bed reactor (CSBR) in an inert atmosphere of non-oxidizing, achieving limonene of 98% purity. Besides, process optimization via pinch analysis has been performed to recover 223.84 kWh of energy using heat integration, resulting in 62.06% of energy saving. From the economic analysis conducted, the total capital investment is RM12.9 million and the annual revenue is RM124.7 million with an annual limonene production of 543.1 tons. The financial analysis demonstrated a robust return on investment (ROI) of 303%, a simple payback period of 0.33 years and ta Net Present Value (NPV) of RM 432.2 million.

Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

In silico characterization of the Limonene synthase gene in citrus species under freezing tolerance

Bitterness in citrus fruit extract is caused by changes and transformations in limonoids and the production of limonene, a bitter-tasting substance. Limonene synthase enzyme is responsible for producing limonene. This study examined the effect of low-temperature stress on the expression of the limonene synthase gene in four different citrus species: Miyagawa mandarin (Citrus unshiu), Thomson Navel Orange (Citrus sinensis var. Thomson navel), Ruby Star grapefruit (Citrus paradisi var. Ruby star), and Lisbon lemon (Citrus limon var. Lisbon). These species were grafted on two-year-old sour orange rootstocks. The experiment was carried out as a factorial in the form of a completely random design. Three temperatures were used for the cold treatment (−8, −4, 4 °C). The expression sequence of the Limonene synthase gene was predicted using bioinformatic software such as PLANT Care and PlantPan. The results showed significant variation in the frequency of amino acids in the LS gene. Leucine (11.36) and glutamic acid (7.57) had the highest frequency. The analysis of the Limonene synthase gene promoter revealed different Cis-element regions such as response elements to light, hormones, and abiotic stresses on the promoter of these genes. The gene promoter had many regions related to binding sites of MYB transcription factors, response regions to hormones, cold (Dehydrin with 15 cis-regulatory elements), and drought, emphasizing the role of this gene in abiotic stress response. The gene expression patterns of the Limonene synthase showed that the highest and lowest expression occurred at − 4 °C in Lisbon lemon (−132.28), Ruby Star grapefruit (−70.06), Thomson Navel Orange (−2.88), and Miyagawa Mandarin (−3.88), confirming the role of this gene in abiotic and biotic stress response.

Machine learning-driven prediction and optimization of pyrolysis oil and limonene production from waste tires

The pyrolysis oil from waste tires possesses significant economic value and is a crucial factor in determining the industrial viability of tire pyrolysis processes. Limonene in pyrolysis oil is a significant component with extremely high industrial application value. In the process of tire pyrolysis, predicting the pyrolysis products through operating conditions and feedstock composition can effectively control industrial operations and enhance operational efficiency. However, there is currently a lack of robust prediction methods for pyrolysis oil and limonene yield. This study proposes the application of machine learning to predict the yield of tire pyrolysis oil and limonene. Artificial Neural Network (ANN) and Random Forest (RF) models were developed to create prediction models. In the statistical analysis, RF achieved optimal R2 median values of 0.83 and 0.64 for pyrolysis oil and limonene predictions during the testing stage.For the prediction of limonene yield, the best R2 and RMSE values in the testing and training stages were 0.844, 3.76, and 0.964, 1.91, respectively. For the prediction of pyrolysis oil yield, the corresponding values were 0.926, 4.1, and 0.985, 1.889, in the testing and training stages, respectively.Temperature was identified as the most critical operating condition affecting pyrolysis oil and limonene yields, with relative importance percentages of 20.6% and 12.2% for oil and limonene yields, respectively. The optimal operating parameter range conducive to limonene yield is as follows: temperature between 350 and 450 °C, residence time between 0 and 50 min, and tire particle size greater than 15 mm.This study lays the foundation for the production of oil and the extraction of limonene from tire pyrolysis.

Volatile Oil Yield and its Components of Menthol and Limonene of Peppermint Affected by Foliar Spray and Lighting Intensity

A field experiment was conducted at a research station in the College of Agricultural Engineering Sciences, University of Baghdad, in spring 2021 to study the effects of foliar spraying of yeast extract, urea, and nano-amino acids and two lighting intensities on volatile oil yield and its components, menthol and limonene. The experiment had two factors: ambient type and lighting. The second component was foliar spraying yeast extract at 5 and 7.5 g.L-1, urea at 1000 and 2000 mg.L-1, and nano amino acids at 1 and 1.5 ml.L-1. P, K, and half of nitrogen were supplied to the soil. Randomized Complete Block Design (RCBD) with three replicates was used for the experiment. Plants grown at 100% lighting intensity had the highest percentage and production of aromatic oil, menthol, and limonene (2.24, 45.49, and 3.30%; 20.20, 9.49, and 0.74 kg.ha-1, respectively) compared to 50% lighting intensity (1.77, 31.00, and 1.91%; 11.64, 3.62, 0.24 kg.ha-1). The two treatments of spraying urea (2000mgN.L-1) and nano amino acids (1.5ml.L-1) at 100% lighting intensity yielded the highest percentage and yield of aromatic oil, menthol, and limonene at 3.07, 45.78, 3.99%; 50.23, 4.60%; 32.74, 16.44, 1.50 kg.ha-1, 29.37, 14.32, 1.17 kg.ha-1, respectively.

The public-health significance of far-UVC-induced indoor ozone and its associated secondary chemistry.

There has been much recent interest in whole-room far-UVC (wavelength around 222 nm) to markedly and safely reduce overall levels of airborne pathogens in occupied indoor locations. Far-UVC light produces very low levels of ozone-in real-world scenarios induced ozone levels of less than 10 ppb, and much less in moderately or well-ventilated rooms compliant with US far-UVC dose recommendations, and very much less in rooms compliant with international far-UVC dose standards. At these very low ozone levels, there is no epidemiological evidence of increased health risks from any of the very large outdoor ozone studies, whether from ozone alone or from ozone plus associated pollutants. Indoors, at the low ozone concentrations of relevance here, ozone does not react rapidly enough with preexisting airborne volatile organic compounds to compete with even extremely low levels of room ventilation, so significant ozone-induced ultrafine particle production is very unlikely. Direct measurements in real-life room scenarios are consistent with these conclusions. A potential exception is the cleaning material limonene, which has an unusually high ozone interaction cross-section; in the far-UVC context, turning off far-UVC lights during cleaning with limonene products would be reasonable.

Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

Sphaerotilus natans hemoglobins have an NADH oxidation activity and promote the yield of limonene in an engineered E. coli strain

Bacterial hemoglobins play important roles inside the cell. Phylogenetically, they belong to three different families: the single domain hemoglobin, flavohemoglobin and truncated hemoglobin. Vitreoscilla hemoglobin (VHb) is the first characterized bacterial hemoglobin, and belongs to the single domain hemoglobin family. Heterologous expression of VHb promotes the growth of host cells under microaerobic conditions, and enhances the yield of products during fermentation. Although VHb has been widely applied in the biotechnology field, other bacterial hemoglobins have not demonstrated similar applications. In this study, we identified four bacterial hemoglobins from the microaerobic growing bacterium Sphaerotilus natans, including one flavohemoglobins (FHB) and three truncated hemoglobins (THB1, THB2 and THB3). Absorption spectrum studies validate the existent of the Soret peak and Q-band characteristic to heme and suggest heme groups in FHB and THB1 are hexa- or penta-coordinated, respectively. Our studies demonstrate that FHB and all three truncated hemoglobins have NADH oxidation and radical production activities, which is surprising since truncated hemoglobins do not have a reductase domain that could bind NADH. However, the M. tuberculosis HbN does not show these activities, indicating they are not universal among truncated hemoglobins. Docking studies suggest the nicotinamide ring of NADH may bind to the distal heme pocket of THB1, suggesting the direct electron transfer from NADH to heme might be possible. Our truncated hemoglobins also show peroxidase activities that in THB2 and THB3 could be inhibited by FdR, indicating possible interactions between FdR and truncate hemoglobins. Expression of FHB and THB1 in E. coli could promote cell growth. THB1 also enhances the production of limonene in an engineered E. coli strain, while VHb does not have this effect, which suggests that studies on truncated hemoglobins may lead to the discovery of new and more powerful tools that could have profound impact on biotechnology.

Terpene produced by coexpression of the TPS and P450 genes from Lavandula angustifolia protects plants from herbivore attacks during budding stages

To deter herbivore attacks, plants employ a diverse array of volatile compounds, particularly during the early developmental stages. The highly expressed genes LaTPS7, LaTPS8, and LaCYP71D582 were identified during the budding phases of Lavandula angustifolia. In vitro studies revealed that LaTPS7 generated nine distinct compounds, including camphene, myrcene, and limonene. LaTPS8 enzymatically converted eight volatiles by utilizing geranyl diphosphate and nerolidyl diphosphate as substrates. Overexpression of plastid-localized LaTPS7 in Nicotiana benthamiana resulted in the production of limonene. Furthermore, the endoplasmic reticulum-associated enzyme LaCYP71D582 potentially converted limonene into carveol. In N. benthamiana, LaTPS8 is responsible for the synthesis of α-pinene and sylvestrene. Furthermore, leaves transfected with LaTPS7 and leaves cotransfected with LaTPS7 and LaCYP71D582 exhibited a repellent effect on aphids, with an approximate rate of 70%. In comparison, leaves with an empty vector displayed a repellent rate of approximately 20%. Conversely, tobacco leaves expressing LaTPS7 attracted ladybugs at a rate of 48.33%, while leaves coexpressing LaTPS7 and LaCYP71D582 attracted ladybugs at a slightly higher rate of 58.33%. Subsequent authentic standard tests confirmed that limonene and carveol repel Myzus persicae while attracting Harmonia axyridis. The promoter activity of LaTPS7 and LaCYP71D582 was evaluated in Arabidopsis thaliana using GUS staining, and it was observed that wounding stimulated the expression of LaTPS7. The volatile compounds produced by LaTPS7, LaTPS8, and LaCYP71D582 play a crucial role in plant defence mechanisms. In practical applications, employing biological control measures based on plant-based approaches can promote human and environmental health.

Optimization of Fermentation Conditions for Elevating Limonene Production with Engineered Rhodosporidium toruloides

Limonene is a valuable monoterpenoid with diverse applications in food, medicine, agriculture, etc. Currently, the commercial production of limonene from citrus pomace cannot meet the evergrowing market demand. With the development of synthetic biology, microbial biosynthesis is offering a sustainable alternative to plant-based extraction. However, the performance of engineered strains can be affected by many factors including the microbial host, the cultivation conditions, etc. Therefore, in the present study, potential factors that influence the limonene production of the engineered strains were investigated through single-factor and orthogonal experiments. The optimized medium was MM, with a working volume of 50 mL, inoculum OD600 = 0.6, 22 °C, pH 6, and a dodecane coverage (v/v) of 20%. The final limonene titer was improved from 52.5 to 358.1 mg/L, a 586% net increase in titer and 34.8-fold improvement in production efficiency (mg/OD600) on 250-mL flasks. The results here demonstrated that R. toruloides could be explored as an efficient limonene producer and could facilitate the study of the biosynthesis of other terpenes using R. toruloides.

Attraction of Pissodes castaneus (Coleoptera, Curculionidae) to Pinus taeda: laboratory and field evaluation

Coniferous trees of the genus Pinus (Pinaceae) are under continuous threats by numerous herbivorous insect species and pathogens attacking nearly all parts and tissues of the plants. To defend themselves, pine trees produce large amounts of oleoresin that is accumulated in a highly developed network of specialized resin ducts, which are distributed in the wood, bark, and needles. Such defense reactions in pines can be induced by the attack of herbivores. The banded pine weevil, Pissodes castaneus (De Geer, 1775) (Coleoptera, Curculionidae), is an important pest of Pinus in Brazil, where it has been an invasive species since 2001. The female lays its eggs under the tree bark of trees and the larvae feed in the phloem of the trunk and branches, interrupting the sap circulation and eventually causing its death. In the present study, we conducted detailed GC–MS analyses of volatiles emitted by twigs of Pinus taeda L. We analyzed how the attack by P. castaneus males and females affects the volatile pattern emitted by the twigs. When comparing volatiles produced by healthy plants and by female- and male-attacked P. taeda, qualitative and quantitative differences were detected, as the decreased production of limonene, germacrene D and (E)-caryophyllene and the increase of α-pinene. Laboratory bioassays showed that plants attacked by male and female P. castaneus were more attractive to the insects. Understanding about what compounds may attract or repel the insects may help in the development of more effective traps, as well as preventing stress to avoid infestation.

The Bph45 Gene Confers Resistance against Brown Planthopper in Rice by Reducing the Production of Limonene.

Brown planthopper (BPH), a monophagous phloem feeder, consumes a large amount of photoassimilates in rice and causes wilting. A near-isogenic line 'TNG71-Bph45' was developed from the Oryza sativa japonica variety 'Tainung 71 (TNG71) carrying a dominant BPH-resistance locus derived from Oryza nivara (IRGC 102165) near the centromere of chromosome 4. We compared the NIL (TNG71-Bph45) and the recurrent parent to explore how the Bph45 gene confers BPH resistance. We found that TNG71-Bph45 is less attractive to BPH at least partially because it produces less limonene. Chiral analysis revealed that the major form of limonene in both rice lines was the L-form. However, both L- and D-limonene attracted BPH when applied exogenously to TNG71-Bph45 rice. The transcript amounts of limonene synthase were significantly higher in TNG71 than in TNG71-Bph45 and were induced by BPH infestation only in the former. Introgression of the Bph45 gene into another japonica variety, Tainan 11, also resulted in a low limonene content. Moreover, several dominantly acting BPH resistance genes introduced into the BPH-sensitive IR24 line compromised its limonene-producing ability and concurrently decreased its attractiveness to BPH. These observations suggest that reducing limonene production may be a common resistance strategy against BPH in rice.

Online SFE-SFC-MS/MS colony screening: A high-throughput approach for optimizing (-)-limonene production

Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.

Metabolic Engineering of the Isopentenol Utilization Pathway Enhanced the Production of Terpenoids in Chlamydomonas reinhardtii.

Eukaryotic green microalgae show considerable promise for the sustainable light-driven biosynthesis of high-value fine chemicals, especially terpenoids because of their fast and inexpensive phototrophic growth. Here, the novel isopentenol utilization pathway (IUP) was introduced into Chlamydomonas reinhardtii to enhance the hemiterpene (isopentenyl pyrophosphate, IPP) titers. Then, diphosphate isomerase (IDI) and limonene synthase (MsLS) were further inserted for limonene production. Transgenic algae showed 8.6-fold increase in IPP compared with the wild type, and 23-fold increase in limonene production compared with a single MsLS expressing strain. Following the culture optimization, the highest limonene production reached 117 µg/L, when the strain was cultured in a opt2 medium supplemented with 10 mM isoprenol under a light: dark regimen. This demonstrates that transgenic algae expressing the IUP represent an ideal chassis for the high-value terpenoid production. The IUP will facilitate further the metabolic and enzyme engineering to enhance the terpenoid titers by significantly reducing the number of enzyme steps required for an optimal biosynthesis.

First in vivo analysis of the regulatory protein CP12 of the model cyanobacterium Synechocystis PCC 6803: Biotechnological implications.

We report the first in vivo analysis of a canonical CP12 regulatory protein, namely the unique CP12 of the model cyanobacterium Synechocystis PCC 6803, which has the advantage of being able to grow photoautotrophically, photomixotrophically, and photoheterotrophically. The data showed that CP12 is dispensable to cell growth under standard (continuous) light and light/dark cycle, whereas it is essential for the catabolism of exogenously added glucose that normally sustains cell growth in absence of photosynthesis. Furthermore, to be active in glucose catabolism, CP12 requires its three conserved features: its AWD_VEEL motif and its two pairs of cysteine residues. Also interestingly, CP12 was found to regulate the redox equilibrium of NADPH, an activity involving its AWD_VEEL motif and its C-ter cysteine residues, but not its N-ter cysteine residues. This finding is important because NADPH powers up the methylerythritol 4-phosphate (MEP) pathway that synthesizes the geranyl-diphosphate (GPP) and farnesyl-diphosphate (FPP) metabolites, which can be transformed into high-value terpenes by recombinant cyanobacteria producing plant terpene synthase enzymes. Therefore, we have introduced into the Δcp12 mutant and the wild-type (control) strain our replicative plasmids directing the production of the monoterpene limonene and the sesquiterpene bisabolene. The photosynthetic production of both bisabolene and limonene appeared to be increased (more than two-fold) in the Δcp12 mutant as compared to the WT strain. Furthermore, the level of bisabolene production was also higher to those previously reported for various strains of Synechocystis PCC 6803 growing under standard (non-optimized) photoautotrophic conditions. Hence, the presently described Δcp12 strain with a healthy photoautotrophic growth and an increased capability to produce terpenes, is an attractive cell chassis for further gene manipulations aiming at engineering cyanobacteria for high-level photoproduction of terpenes.

Production of limonene from waste tires via catalytic fast pyrolysis: a statistical–based screening on Ni-, Pd-, Co-, and Fe-supported catalysts

Production of limonene from waste tires via catalytic fast pyrolysis: a statistical–based screening on Ni-, Pd-, Co-, and Fe-supported catalysts