Triterpene Hydrocarbons Research Articles

Recent advances in the microbial production of squalene.

Squalene is a triterpene hydrocarbon, a biochemical precursor for all steroids in plants and animals. It is a principal component of human surface lipids, in particular of sebum. Squalene has several applications in the food, pharmaceutical, and medical sectors. It is essentially used as a dietary supplement, vaccine adjuvant, moisturizer, cardio-protective agent, anti-tumor agent and natural antioxidant. With the increased demand for squalene along with regulations on shark-derived squalene, there is a need to find alternatives for squalene production which are low-cost as well as sustainable. Microbial platforms are being considered as a potential option to meet such challenges. Considerable progress has been made using both wild-type and engineered microbial strains for improved productivity and yields of squalene. Native strains for squalene production are usually limited by low growth rates and lesser titers. Metabolic engineering, which is a rational strain engineering tool, has enabled the development of microbial strains such as Saccharomyces cerevisiae and Yarrowia lipolytica, to overproduce the squalene in high titers. This review focuses on key strain engineering strategies involving both in-silico and in-vitro techniques. Emphasis is made on gene manipulations for improved precursor pool, enzyme modifications, cofactor regeneration, up-regulation of limiting reactions, and downregulation of competing reactions during squalene production. Process strategies and challenges related to both upstream and downstream during mass cultivation are detailed.Graphical abstract

Molecular basis of triterpene-based chemophenetics in ferns

A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.

Identification and transmission of phytoplasmas and their impact on essential oil composition in Aerva javanica.

Aerva javanica plants showing symptoms of witches' broom, little leaf, and leaf roll were observed in 50% of the A. javanica plants grown in the deserts of Hormozgan province, Iran. This study was carried out to verify possible phytoplasma association with this disease, insect vector, and modification of the chemical composition of symptomatic A. javanica. Aerial parts were collected at the full flowering stage from symptomatic and asymptomatic plants and samples were examined for phytoplasma presence by nested-PCR assays. PCR and sequencing analysis showed that a phytoplasma was associated with the disease sharing 99% sequence identity with 'Candidatus Phytoplasma aurantifolia'-related strains (16SrII). Furthermore, this phytoplasma was transmitted to healthy periwinkle plants under experimental conditions by the leafhopper Austroagallia sinuata that was then demonstrated to be a vector of this phytoplasma. Analysis of the oils carried out by GC-MS revealed the enrichment of the triterpene hydrocarbons (2.3% in asymptomatic vs. 11.8% in symptomatic samples) and the hydrocarbons (69.3% vs. 85.93%) as compared to the abundance in asymptomatic samples. Conversely, a matching decrease in ketone (3.51% vs. 0.6%) was observed in the symptomatic plants. These results indicate that phytoplasma presence is influencing the secondary metabolites production in infected plants.

Genetic modification of the thraustochytrid Aurantiochytrium sp. 18W-13a for cellobiose utilization by secretory expression of β-glucosidase from Aspergillus aculeatus

Thraustochytrids are osmoheterotrophic protists that are emerging as a promising group of oleaginous microorganisms due to their high DHA, EPA and palmitic acid production. Squalene, a triterpene hydrocarbon known for its antioxidant, antitumorigenic and emollient properties, is accumulated in some strains of thraustochytrids especially in the genus Aurantiochytrium. Thraustochytrids can produce extracellular enzymes to degrade organic matter as their means of nutrition. Many studies attempt to grow thraustochytrids in cost-effective substrates such as lignocellulosic biomass. As the first step towards cellulose utilization, we genetically modified Aurantiochytrium sp. strain 18W-13a to express and secrete a highly active cellulase, β-glucosidase from Aspergillus aculeatus (AaBgl). The transformant strain, AaBgl+, exhibited increasing growth and enzyme activity under cellobiose as the sole carbon source. Native PAGE zymogram showed an active β-glucosidase enzyme in the supernatant of the AaBgl+ strain. Both wild-type and AaBgl+ strains can produce squalene and fatty acids DHA and DPA. This work is one of the first reports in targeted secretion of a functional enzyme in thraustochytrids. Moreover, enhanced expression of cellulases has never been done in thraustochytrids. We hope that this study could pave way for establishing consolidated bioprocessing thraustochytrid strains.

Cucurbitacin B Induces the Lysosomal Degradation of EGFR and Suppresses the CIP2A/PP2A/Akt Signaling Axis in Gefitinib-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients carrying an epidermal growth factor receptor (EGFR) mutation are initially sensitive to EGFR-tyrosine kinase inhibitors (TKIs) treatment, but soon develop an acquired resistance. The treatment effect of EGFR-TKIs-resistant NSCLC patients still faces challenges. Cucurbitacin B (CuB), a triterpene hydrocarbon compound isolated from plants of various families and genera, elicits anticancer effects in a variety of cancer types. However, whether CuB is a viable treatment option for gefitinib-resistant (GR) NSCLC remains unclear. Here, we investigated the anticancer effects and underlying mechanisms of CuB. We report that CuB inhibited the growth and invasion of GR NSCLC cells and induced apoptosis. The inhibitory effect of CuB occurred through its promotion of the lysosomal degradation of EGFR and the downregulation of the cancerous inhibitor of protein phosphatase 2A/protein phosphatase 2A/Akt (CIP2A/PP2A/Akt) signaling axis. CuB and cisplatin synergistically inhibited tumor growth. A xenograft tumor model indicated that CuB inhibited tumor growth in vivo. Immunohistochemistry results further demonstrated that CuB decreased EGFR and CIP2A levels in vivo. These findings suggested that CuB could suppress the growth and invasion of GR NSCLC cells by inducing the lysosomal degradation of EGFR and by downregulating the CIP2A/PP2A/Akt signaling axis. Thus, CuB may be a new drug candidate for the treatment of GR NSCLC.

Docking analysis of models for 4-hydroxy-3-methylbut-2-enyl diphosphate reductase and a ferredoxin from Botryococcus braunii, race B.

The green microalga Botryococcus braunii Showa, which produces large amounts of triterpene hydrocarbons, exclusively uses the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosyntheses, and the terminal enzyme in this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), is regarded as a light-dependent key regulatory enzyme. In order to investigate the possible association of HDR and ferredoxin in this organism, we constructed tertiary structure models of B. braunii HDR (BbHDR) and one of ferredoxin families in the alga, a photosynthetic electron transport F (BbPETF)-like protein, by using counterparts from E. coli and Chlamydomonas reinhardtii as templates, respectively, and performed docking analysis of these two proteins. After docked models are superimposed onto their counterpart proteins in a non-photosynthetic organism, Plasmodium falciparum, the BbPETF-like protein comes in contact with the backside of BbHDR, which was defined in a previous report (Rekittke et al. 2013), and the distance of the two Fe-S centers is 14.7 Å. This distance is in almost the same level as that for P. falicarum, 12.6 Å. To our knowledge, this is the first model suggesting the possible association of HDR with a ferredoxin in O2-evolving photosynthetic organisms.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Component composition of volatile organic substances of dog-rose petals

Purpose. To analyse the qualitative and quantitative composition of volatile organic substances (VOS) in the petals of six species of dog-rose in the collection of M. M. Gryshko National Botanic Garden of the NAS of Ukraine for further bree­ding.Methods. Volatile organic substances were obtained by steam distillation. VOS was determined using gas chromato­graphy (Chromatograph Agilent Technologies 6890).Results. The qualitative and quantitative composition of volatile organic substances in the petals in such species as Rosa L. (R. roullettii HCh (Correvon), R. multiflora Thunb., R. pimpinellifolia L., R. canina L., R. centifolia L., R. rugosa Thunb.) was investigated. According to the results of the research, 105 VOS were discovered in the dog-rose petals, among which 11 were not identified. The dog-rose petals of identified VOC contained 16 components which share exceeds 5%, 31 – was ranging from 1% to 5%, 47 – was less than 1%. They belonged to different groups including monoterpenoids, sesquiterpenoids, sesquiterpene alcohols, saturated unbranched hydrocarbons etc. The largest number of components were identified in the petals of R. roulettii (49) and R. centifolia (45), while the number of identified components in species R. multiflora (33), R. rugosa (31) and R. canina (30) was relatively smaller. All studied dog-rose species were characterized by the presence of such saturated unbranched hydrocarbons as decan, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nanodecane, heneicosane, tricosane, tetracosane, pentacosane, heptocosane, hentriacontane. Triterpene hydrocarbon squalene is the important component of the VOS complex in dog-rose pen tals. b-phenylethyl alcohol to be one of the main components of the rose essential oils was found during investigation in the following four dog-rose species as R. centifolia (0.61%), R. pimpinellifolia (3.56%), R. rugosa (4.24%), and R. multiflora (5.43%). Significant content of dihydro-b-ionol (18.46%), dihydro-b-ionone (0.69%), thias­piran A (1.35%), and thiaspiran B (2.17%) were revealed in the petals of R. roulettii, which also have an influence on the aromatic bouquet of the rose oil.Conclusions. For the first time, 105 volatile organic substances were found in the petals of six species of dog-rose, 11 of which were not identified. It was found out that the largest number of components were identified in the petals of R. roulettii (49) and R. centifolia (45). The number of VOS identified in species R. multiflora (33), R. rugosa (31), and R. canina (30) was relatively smaller. Component composition of VOS in the petals of studied dog-rose species is an important constituent in breeding investigations of prospective producers for the various purposes of use.

Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race

The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a km for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4μM, and a kcat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae.

Raman spectra and DFT calculations for botryococcene and methylsqualene hydrocarbons from the B race of the green microalga Botryococcus braunii

Botryococcus braunii, a green colonial microalga, is a prodigious producer of liquid hydrocarbon oils that can be used as renewable feedstocks for producing combustion engine fuels. The B race of B. braunii mainly produces the triterpene hydrocarbons known as botryococcenes, which have over twenty known structures. Minor hydrocarbons in the B race include the triterpene methylsqualenes. Here we report an examination of the molecular structure for ten botryococcenes and five methylsqualenes using Raman spectroscopy and density functional theory (DFT) calculations in an effort to distinguish between these structurally similar molecules by spectroscopic approaches. The DFT calculations show that these molecules have between 243 and 271 vibrational frequencies. A comparison of the experimental Raman spectroscopy and DFT calculations indicates several spectral regions such as those for ν(CC) stretching, CH2/CH3 bending, and ring bending can be used to distinguish between these molecules. In an extension of this analysis, a broadband coherent anti-Stokes Raman spectroscopy (BCARS) analysis was used to clearly distinguish between several botryococcenes isomers.

Utilization of ammonium by the hydrocarbon-producing microalga, Botryococcus braunii Showa

As a prospective feedstock for sustainable biofuels, the green microalga Botryococcus braunii race B has attracted significant attention since this colony-forming alga has the ability to accumulate considerable amounts of triterpene hydrocarbons, botryococcenes, in its extracellular matrix. However, its primary metabolism, nutrient uptake and utilization relevant to growth, are still poorly understood in this alga. From this perspective, we investigated the utilization of ammonium by B. braunii as a more reduced form of nitrogen source compared to nitrate, which is generally used for cultures of the alga. Isotope-ratio mass spectrometry analyses indicated that the uptake rate of ammonium was higher than that of nitrate at a certain concentration. When ammonium was added to culture medium, however, it inhibited algal growth even in the presence of nitrate. Buffered culture media ameliorated the toxicity of ammonium, and the alga accumulated the same levels of hydrocarbons and secondary carotenoids, irrespective of nitrogen source. To characterize utilization of ammonium by B. braunii at the molecular level, putative ammonium transporter (AMT) genes were screened from a cDNA library and four isogenes (BbAMT1;1, 1;2, 1;3 and 1;4) were cloned. Two of them, BbAMT1;1 and 1;2, were upregulated under nitrogen deficient conditions. BbAMT1;1 complemented the growth of an ammonium uptake-defective yeast strain, showing it to be a functional gene coding for AMT. These results could pave the way for culturing B. braunii more efficiently using ammonium as a sole nitrogen source by improving ammonium uptake through our understanding of AMT.

Effects of 2-azahypoxanthine on extracellular terpene accumulations by the green microalga Botryococcus braunii, race B

The B race of the green microalga Botryococcus braunii accumulates large amounts of triterpene hydrocarbons that are attractive as a source for biofuels. The alga, however, exhibits rather slow growth and this characteristic has hindered its practical application for biofuel production. In order to test a phytohormone-like compound that may regulate the growth or hydrocarbon production of this alga, the effects of 2-azahypoxanthine (AHX) on algal biomass and terpene contents in the extracellular matrix (ECM) in the Showa strain of B. braunii were investigated. The biomass of the Showa strain increased with AHX supplementation under static culture conditions although such effects were not observed in cultures with aeration or agitation. AHX supplementation significantly induced the accumulation of secondary carotenoids, including species-specific tetramethylsqualene (TMS)-conjugated carotenoids, botryoxanthin A and braunixanthin 1. Conversely, the addition of AHX showed a tendency to slightly lower the triterpene hydrocarbon (botryococcenes) content in the ECM. These metabolic changes in tri- and tetraterpene accumulation could be caused by a reduction in nitrogen uptake from the culture medium due to AHX addition. Thus, there is a possibility that AHX could be used as a reagent to modulate terpene biosynthesis in the B race of B. braunii.

Triterpene hydrocarbon production engineered into a metabolically versatile host--Rhodobacter capsulatus.

Triterpene hydrocarbon biosynthesis of the ancient algae Botryococcus braunii was installed into Rhodobacter capsulatus to explore the production of C30 hydrocarbon in a host capable of diverse growth habits-utilizing carbohydrate, sunlight or hydrogen (with CO2 fixation) as alternative energy feedstocks. Engineering an enhanced MEP pathway was also used to augment triterpene accumulation. Despite dramatically different sources of carbon and reducing power, nearly the same level of botryococcene or squalene (∼5 mg oil/g-dry-weight [gDW]) was achieved in small-scale aerobic heterotrophic, anaerobic photoheterotrophic, and aerobic chemoautotrophic growth conditions. A glucose fed-batch bioreactor reached 40 mg botryococcene/L (∼12 mg/gDW), while autotrophic bioreactor performance with CO2 , H2 , and O2 reached 110 mg/L (16.7 mg/gDW) during batch and 60 mg/L (23 mg/gDW) during continuous operation at a dilution rate corresponding to about 10% of μ(max). Batch and continuous autotrophic specific productivity was found to reach 0.5 and 0.32 mg triterpene/g DW/h, comparable to prior reports for terpene production driven by heterotrophic growth conditions. This demonstrates the feasibility of alternative feedstocks and trophic modes to provide comparable routes to biochemicals that do not rely on sugar.

Isolation and characterization of two squalene epoxidase genes from Botryococcus braunii, race B.

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.

Analysis of diterpenes and triterpenes from plant foliage and roots.

Terpene specialized metabolites exhibit multiple functions in plant-environment interactions and plant development. Molecular biologists investigating the biochemistry and molecular function of terpenes need to apply robust but yet sensitive analytical methods optimized and adapted to the structural diversity and often varying concentrations of terpene compounds in plant tissues. Here we present hands-on protocols for sample preparation and GC-MS or LC-MS/MS analysis of selected diterpene and triterpene hydrocarbons or oxygenated derivatives from roots and shoots of Arabidopsis and rice.

Hydrocarbon production in high density Botryococcus braunii race B continuous culture

Continuous cultures of Botryococcus braunii race B were maintained at photosynthetic cell densities as high as 20 g dry weight per liter for up to 3 months. Growth associated triterpene hydrocarbon accumulation was nearly constant at 22.5% of dry weight for a range of growth rates maintained by daily replacement of 5-15% of the respective cultures. The ability to achieve high cell concentrations and oil levels of roughly 5 g triterpene oil/L resulted from a combination of high light (∼ 1/4 full sun for 15 h/day) and replenishing stoichiometrically balanced growth medium. Due to light-limited growth conditions, cell concentration dropped nearly linearly with increased dilution rate. This reduction in cell number resulted in increased productivity per cell at higher dilution rates and was accompanied by a dramatic increase in algae colony size from 0.09 to 0.343 mm at high dilution rate. This change in colony size resulted in an equally dramatic change in optical density (OD) per gram dry weight, which precluded use of simple correlations of OD and cell concentration. A trickle-film photobioreactor was also demonstrated as a scalable approach to achieving these ultra-high cell concentrations. Additional media analysis revealed a steady increase in photobioreactor conductivity suggesting an accumulation of ions may be the reason for rapid culture crash and washout observed at all dilution rates after several months of continuous operation. The volumetric productivity of 22.5 mg oil/L/photo-h reported here is more than an order of magnitude higher than previous reports for B. braunii race B, reflecting the high cell densities used in this work and substantiating a higher metabolic rate for B. braunii race B than previously surmised from its relatively long doubling times.

Catalytic conversion of Botryococcus braunii oil to diesel fuel under mild reaction conditions

Although the non-oxygenated triterpenic hydrocarbons (primarily C34H58) produced by the Bot-22 strain of Botryococcus braunii are exceptionally promising candidates for automobile fuel applications, it remains necessary to develop appropriate cracking technologies to convert these oils into a fuel with the required properties. The aim of this research was to develop an on-site process capable of converting the oil extracted from Bot-22 (Bot-oil) to a fuel that satisfies the specifications for diesel fuels, a so-called “drop-in” fuel. One of our primary goals for this on-site conversion was to have it operate as a low-temperature process. In this study, an experimental analysis of Bot-oil catalytic conversion under mild conditions was performed. The results demonstrated that the Bot-oil conversion reaction will proceed at temperatures as low as approximately 200 °C, and that the lowest temperature which resulted in a suitably efficient reaction was found to be 260 °C, generating a yield of 85 %. The physical properties of the converted oil were found to come close to satisfying Japan’s JIS #2 diesel fuel specification. The converted oil’s cetane number (CN), one of the most important quality indexes of diesel fuels, was estimated by measuring its ignition delay in a constant-volume combustion chamber. The estimated CN of the converted oil had a value of 40, which is 5 points below the JIS #2 requirement. It is thought that the Bot-oil can be used to produce a drop-in fuel using a simple on-site conversion process, although further development is required to achieve this aim.

Algal biofuels

The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO2 concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO2 by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.

The single cellular green microalga Botryococcus braunii, race B possesses three distinct 1-deoxy-d-xylulose 5-phosphate synthases

Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy-d-xylulose 5-phosphate synthase (DXS, EC 2.2.1.7). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.

Physical Properties of Hydrocarbon Oils Produced by Botryococcus Braunii: Density, Kinematic Viscosity, Surface Tension, and Distillation Properties

In automotive engine combustion, particularly in case of compression ignition in diesel engines, physical properties of the fuel have a direct impact on the spray combustion process. The density, kinematic viscosity, surface tension, and distillation properties of triterpenic hydrocarbons extracted from the BOT-22 strain of Botryococcus braunii (called Bot-oil) were measured, and these properties were compared with those of standard diesel fuel. The density and surface tension of Bot-oil were almost the same as those of the diesel fuel. However, the kinematic viscosity of Bot-oil was 55 to 58 cSt at room temperature, which was 16 times higher than that of the diesel fuel. In addition, the distillation temperature of Bot-oil was approximately 430 °C, which was over 100 °C higher than that of the diesel fuel. To attain the properties required for fuel, a cracking technology for Bot-oil must be established.