Effect of two ergosterol biosynthesis inhibitors on lycopene production by Blakeslea trispora

Limiting ergosterol accumulation through metabolic control increased lycopene production by Blakeslea trispora. Lycopene and ergosterol are both biosynthesized from a common precursor, farnesyl diphosphate (FPP). The effects of two ergosterol biosynthesis inhibitors, terbinafine hydrochloride (TH) and ketoconazole, on the production of lycopene by B. trispora were investigated. TH at 0.7 mg/l and ketoconazole at 30 mg/l added to the medium at 48 h of fermentation caused an increase in lycopene content of 23% or 277%, respectively. The timing of addition for both inhibitors at 48 h resulted in the most optimal lycopene productivity, however, compared with TH, ketoconazole was superior in enhancing lycopene production by inhibiting ergosterol biosynthesis.     

Improved production of lycopene and β-carotene by Blakeslea trispora with oxygen-vectors

Lycopene and β-carotene production were increased when oxygen-vectors, n-hexane and n-dodecane, were added to cultures of Blakeslea trispora because of the enhanced dissolved oxygen concentrations. With 1% (v/v) n-hexane or n-dodecane added in the medium, lycopene production was 51% or 78% higher and β-carotene production was 44% or 65% higher than that of the control, respectively. The highest lycopene and β-carotene production, 533 mg l⁻¹and 596 mg l⁻¹, were obtained when 1% (v/v) n-dodecane and 0.1% (w/v) Span 20 were added together, which were 2.1-fold and 1.8-fold of the control, respectively.     

Carotenoid synthesis and phytoene synthase activity during mating of Blakeslea trispora

Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (−) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional phytoene synthase and lycopene cyclase.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [¹⁴C]acetyl-CoA, L-[¹⁴C]leucine, or R-[¹⁴C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Ubiquinone and carotene production in the <Emphasis Type="Italic">Mucorales Blakeslea</Emphasis> and <Emphasis Type="Italic">Phycomyces</Emphasis>

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of β-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.     

Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis

As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statistically-based experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe³⁺, Co²⁺, Mn²⁺, Al²⁺ and Zn²⁺ (within the range 0–50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (β-CAR, β-carotene; γ-CAR, γ-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant (P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, β-CAR, γ-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 μg/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; β-CAR = 50.3%; γ-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.     

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24 h and then shifted to 5.0 after 24 h. Using this strategy, the highest β-carotene production (50.39 mg/l) and the formation rate (1.40 mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H₂O₂ enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.     

24-Methylenecyclopropane steroidal inhibitors: A Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei

A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15 ± 5 μM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24β-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (k_cat/k_inact = 0.13 min^− 1/0.08 min^− 1; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC–MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases.     

Metabolic Regulation of Trisporic Acid on Blakeslea trispora Revealed by a GC-MS-Based Metabolomic Approach

The zygomycete Blakeslea trispora is used commercially as natural source of â-carotene. Trisporic acid (TA) is secreted from the mycelium of B. trispora during mating between heterothallic strains and is considered as a mediator of the regulation of mating processes and an enhancer of carotene biosynthesis. Gas chromatography-mass spectrometry and multivariate analysis were employed to investigate TA-associated intracellular biochemical changes in B. trispora. By principal component analysis, the differential metabolites discriminating the control groups from the TA-treated groups were found, which were also confirmed by the subsequent hierarchical cluster analysis. The results indicate that TA is a global regulator and its main effects at the metabolic level are reflected on the content changes in several fatty acids, carbohydrates, and amino acids. The carbon metabolism and fatty acids synthesis are sensitive to TA addition. Glycerol, glutamine, and ã-aminobutyrate might play important roles in the regulation of TA. Complemented by two-dimensional electrophoresis, the results indicate that the actions of TA at the metabolic level involve multiple metabolic processes, such as glycolysis and the bypass of the classical tricarboxylic acid cycle. These results reveal that the metabolomics strategy is a powerful tool to gain insight into the mechanism of a microorganism’s cellular response to signal inducers at the metabolic level.     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Molecular cloning and functional expression of two key carotene synthetic genes derived from Blakeslea trispora into E. coli for increased β-carotene production

Blakeslea trispora is used commercially to produce β-carotene. Isopentenyl pyrophosphate isomerase (IPI) and geranylgeranyl pyrophosphate synthase (GGPS) are key enzymes in the biosynthesis of carotenoids. The cDNAs of genes ipi and carG were cloned from the fungus and expressed in Escherichia coli. Greater GGPS activity was needed in the engineered E. coli when IPP activity was increased. The introduction of GGPS and IPI increased the β-carotene content in E. coli from 0.5 to 0.95?mg/g dry wt.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.