Β-carotene Production Research Articles

2A-linked bi-, tri-, and quad-cistrons for the stepwise biosynthesis of β-carotene, zeaxanthin, and ketocarotenoids in rice endosperm

Foot-and-mouth disease virus (FMDV) 2A constructs have been successfully used for the production of “Golden Rice”, a β-carotene producing rice strain. However, to allay public fears and opposition to plants carrying a mammalian pathogenic viral sequence, 2A-like synthetic sequences from Thosea asigna virus and Infectious myonecrosis virus were used to coordinate the coexpression of carotenoid biosynthetic genes. Here, up to four carotenogenic genes encoding PSY, CRTI, BCH and BKT were concatenated and produced β-carotene, zeaxanthin, and ketocarotenoids (astaxanthin and adonixanthin) in transgenic rice seeds displaying color variation due to the difference in carotenoid content and composition.

Natural β-Carotene Production by Blakeslea trispora Cultivated in Spanish-Style Green Olive Processing Wastewaters.

In the current research, the potential of Spanish-style green olive processing wastewaters (lye and washing waters) exploitation toward natural β-carotene production by Blakeslea trispora was tested for the first time. Mating culture generated by the joint cultivation of the heterothallic fungal strains ATCC 14271 and 14272 in the non-sterile lye and washing waters was able to grow, achieving the phytotoxic hydroxytyrosol degradation by 57.3% and 66.8%, respectively. However, the low sugar and nitrogen content of the streams did not favor carotenogenesis. Alternatively, in the nutrient-enriched effluents, a notable quantity of β-carotene was produced, accounted for 61.2 mg/L (lye) and 64.1 mg/L (washing waters) (82–88% of total carotenoid content). Above all, enriched streams had a noteworthy stimulating effect on the β-carotene synthesis, because both the maximum β-carotene yield per volume of enriched effluents and specific β-carotene production rate were higher when compared with the respective values obtained from trials with synthetic reference medium without added effluents. Hydroxytyrosol and tyrosol showed high stability during the non-sterile process for β-carotene production by B. trispora grown in the enriched effluents. This finding strengthens the potential toward the generation of multiple high-value products, which could lower the natural β-carotene production costs.

Enhanced Lutein Production in Chlamydomonas reinhardtii by Overexpression of the Lycopene Epsilon Cyclase Gene.

Chlamydomonas reinhardtii is a well-established microalgal model species with a shorter doubling time, which is a promising natural source for the efficient production of high-value carotenoids. In the microalgal carotenoid biosynthetic pathway, lycopene is converted either into β-carotene by lycopene β-cyclase or into α-carotene by lycopene ε-cyclase (LCYE) and lycopene β-cyclase. In this study, we overexpressed the LCYE gene in C. reinhardtii to estimate its effect on lycopene metabolism and lutein production. Chlamydomonas transformants (CrLCYE#L1, #L5, and #L6) produced significantly increased amounts of lutein per culture (up to 2.6-fold) without a decrease in cell yields. Likewise, the expression levels of LCYE gene in transformants showed a significant increase compared with that of the wild-type strain. These results suggest that LCYE overexpression enhances the conversion of lycopene to α-carotene, which in turn improves lutein productivity. Interestingly, their β-carotene productivity appeared to increase slightly rather than decrease. Considering that the inhibition of the lycopene cyclization steps often induces higher expression in genes upstream of metabolic branches, this result implies that the redirection from β-carotene to α-carotene by LCYE overexpression might also enhance upstream gene expression, thereby leading to auxiliary β-carotene production.

ROS Induce β-Carotene Biosynthesis Caused by Changes of Photosynthesis Efficiency and Energy Metabolism in Dunaliella salina Under Stress Conditions.

The unicellular alga Dunaliella salina is regarded as a promising cell factory for the commercial production of β-carotene due to its high yield of carotenoids. However, the underlying mechanism of β-carotene accumulation is still unclear. In this study, the regulatory mechanism of β-carotene accumulation in D. salina under stress conditions was investigated. Our results indicated that there is a significant positive correlation between the cellular ROS level and β-carotene content, and the maximum quantum efficiency (Fv/Fm) of PSII is negatively correlated with β-carotene content under stress conditions. The increase of ROS was found to be coupled with the inhibition of Fv/Fm of PSII in D. salina under stress conditions. Furthermore, transcriptomic analysis of the cells cultivated with H2O2 supplementation showed that the major differentially expressed genes involved in β-carotene metabolism were upregulated, whereas the genes involved in photosynthesis were downregulated. These results indicated that ROS induce β-carotene accumulation in D. salina through fine-tuning genes which were involved in photosynthesis and β-carotene biosynthesis. Our study provided a better understanding of the regulatory mechanism involved in β-carotene accumulation in D. salina, which might be useful for overaccumulation of carotenoids and other valuable compounds in other microalgae.

Nitrogen Deficiency Maximizes the Production and Accumulation of Β-Carotene via Induction of Different Macromolecule Derivatives in Dunaliella salina (Dunal) Teodoresco

THIS STUDY aims at investigating the effects of nitrogen deficiency and NaCl stress on β-carotene production and growth of Dunaliella salina (Dunal) Teodoresco. D. salina was exposed to different levels of N: 2.5, 4.9, 7.4 and 9.9mM KNO3 or Na+: 1.3, 1.7, 2.6, 3.4 and 4.3M NaCl. β-carotene production and accumulation were significantly better in nitrogen deficiency-treated D. salina than that in NaCl-treated cells, with a maximum increase of 5.5mg g DW-1 at 2.5mM KNO3. Algal growth, in terms of cell number and the relative growth rate (RGR), was increased with the increase in the availability of nitrogen but decreased by NaCl stress. In contrast, the production of β-carotene by D. salina was enhanced under nitrogen deficiency and excessive salinity, suggesting the opposite effect of nitrogen availability and stressful NaCl on the production and accumulation of β-carotene concomitant with the inhibition of algal growth. GC analysis confirmed that nitrogen deficiency-treated D. salina accumulates β-carotene higher than untreated cells (control), suggesting the great potential of nitrogen deficiency-treated D. salina to produce different β-carotene macromolecule derivatives compared to those in the control. Such different hydrocarbons could be converted into safe β-carotene. Therefore, D. salina could be used as a reliable biological system for β-carotene production. The study recommended cultivating D. salina under low salinity to obtain D. salina cells, but high salinity to obtain their β-carotene.

Накопление нейтральных липидов и β-каротина дрожжами Yarrowia lipolytica на среде с сахарозой в качестве источника углерода

Recombinant Yarrowia lipolytica yeast has been used as a host strain for the expression of the Saccharomyces cerevisiae ScSUC2 gene and Y. lipolytica YlHXK1 gene encoding invertase and hexakinase, respectively. The expression was carried out on the background of the enhanced pathway of the synthesis of neutral lipids. This allowed the yeast to efficiently utilize glucose as a single carbon source. The engineered strain accumulated neutral lipids in amount of 50.7% of biomass dry weight, when cultured in tubes in minimal medium with nitrogen limitation and high sucrose content. The next metabolic engineering step was the use of the CRISPR-Cas9 editing system to introduce a heterologous β-carotene synthesis pathway by the expression of the Mucor circinelloides genes CarRP and CarB, encoding the bifunctional enzyme phytoene synthase/licopene-β-cyclase (carRP) and phytoene dehydrogenase (carB), as well as an increase in the expression level of the Y. lipolytica YlGGS1 gene, encoding geranyl diphosphate synthase enzyme. The β-carotene production on a sucrose-containing medium was shown for the first time; it amounted for 24.0 mg/g dry biomass, or 406.9 mg/L, on the 5th day of cultivation in tubes in a rich medium with a sucrose portioned supply (50 g/L). The corresponding values for a rich medium with a higher sucrose concentration (90 g/L) were 21.9 mg/g dry biomass and 625.8 mg/L. Yarrowia lipolytica, β-carotene, sucrose, CRISPR-Cas9 The plasmids pdKu70Yl-URA3, pLTet-SP-CAT, pARS-Cre-reverse, pMW-att-Cm and pGPD1Yl were kindly provided by Ph.D. I.A. Laptev ("Kurchatov Institute"-GOSNIIGENETIKA NRC). The work was carried out using the equipment of the Multipurpose Scientific Facility of the "Russian State Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»-GOSNIIGENETIKA. The work was supported by state assignment no. 14 of 25.07.2012 AAAA-A20-120093090016-9. The quantitative analysis of lipids and β-carotene was carried out by V.A. Zolottsev within the framework of the "Long-Term Program of Fundamental Scientific Research in the Russian Federation (2021-2030)".

Seco-type β-Apocarotenoid Generated by β-Carotene Oxidation Exerts Anti-inflammatory Effects against Activated Macrophages.

β-Apocarotenoids are the cleavage products of β-carotene. They are found in plants, carotenoid-containing foods, and animal tissues. However, limited information is available regarding the health benefits of β-apocarotenoids. Here, we prepared seco-type β-apocarotenoids through the chemical oxidation of β-carotene and investigated their anti-inflammatory effects against activated macrophages. Oxidation of β-carotene with potassium permanganate produced seco-β-apo-8'-carotenal, in which one end-group formed an "open" β-ring and the other was cleaved at the C-7',8' position. In lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells, seco-β-apo-8'-carotenal inhibited the secretion and mRNA expression of inflammatory mediators such as nitric oxide, interleukin (IL)-6 and IL-1β, and monocyte chemoattractant protein-1. Furthermore, seco-β-apo-8'-carotenal suppressed phosphorylation of c-Jun N-terminal kinase and the inhibitor of nuclear factor (NF)-κB as well as the nuclear accumulation of NF-κB p65. Notably, since seco-β-apo-8'-carotenal exhibited remarkable anti-inflammatory activity compared with β-apo-8'-carotenal, its anti-inflammatory action could depend on the opened β-ring structure. These results suggest that seco-β-apo-8'-carotenal has high potential for the prevention of inflammation-related diseases.

Photosynthetic profiling of a Dunaliella salina mutant DS240G-1 with improved β-carotene productivity induced by heavy ions irradiation

Photosynthetic profiling of a Dunaliella salina mutant DS240G-1 with improved β-carotene productivity induced by heavy ions irradiation

Redirecting Metabolic Flux towards the Mevalonate Pathway for Enhanced β-Carotene Production in M. circinelloides CBS 277.49.

Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of β-carotene production in M. circinelloides strain 277.49 was achieved using response surface methodology (RSM). Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance β-carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of β-carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior β-carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the β-carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10 μg/mL cerulenin, and 150 mg/L ketoconazole, producing maximum β-carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium (1.68 mg/L, 0.17 mg/g). So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced β-carotene production in CBS 277.49.

Metabolic engineering of Saccharomyces cerevisiae for production of β-carotene from hydrophobic substrates.

ABSTRACTβ-Carotene is a yellow–orange–red pigment used in food, cosmetics and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose (YPD) medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained in yeast nitrogen base (YNB) medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

Multiplicity of carotene patterns derives from competition between phytoene desaturase diversification and biological environments

Phytoene desaturases catalyse from two to six desaturation reactions on phytoene, generating a large diversity of molecules that can then be cyclised and produce, depending on the organism, many different carotenoids. We constructed a phylogenetic tree of a subset of phytoene desaturases from the CrtI family for which functional data was available. We expressed in a bacterial system eight codon optimized CrtI enzymes from different clades. Analysis of the phytoene desaturation reactions on crude extracts showed that three CrtI enzymes can catalyse up to six desaturations, forming tetradehydrolycopene. Kinetic data generated using a subset of five purified enzymes demonstrate the existence of characteristic patterns of desaturated molecules associated with various CrtI clades. The kinetic data was also analysed using a classical Michaelis–Menten kinetic model, showing that variations in the reaction rates and binding constants could explain the various carotene patterns observed. Competition between lycopene cyclase and the phytoene desaturases modified the distribution between carotene intermediates when expressed in yeast in the context of the full β-carotene production pathway. Our results demonstrate that the desaturation patterns of carotene molecules in various biological environments cannot be fully inferred from phytoene desaturases classification but is governed both by evolutionary-linked variations in the desaturation rates and competition between desaturation and cyclisation steps.

Phytoene and phytofluene overproduction by Dunaliella salina using the mitosis inhibitor chlorpropham

The halotolerant chlorophyte microalga, Dunaliella salina, is one of the richest sources of carotenoids and will accumulate up to 10% of the dry biomass as β-carotene, depending on the integrated amount of light to which the alga is exposed during a division cycle. Red light also stimulates β-carotene production, as well as increases the 9-cis β-carotene/all-trans β-carotene ratio. In this paper we investigated the effects of chlorpropham (Iso-propyl-N (3-chlorophenyl) carbamate, CIPC), with and without red light, on carotenoid accumulation. Chlorpropham is a well-known carbamate herbicide and plant growth regulator that inhibits mitosis and cell division. Chlorpropham arrested cell division and induced the massive accumulation of colourless phytoene and phytofluene carotenoids, and, to a much lesser extent, the coloured carotenoids. The chlorophyll content also increased. When phytoene per cell accumulated to approximately the same level with chlorpropham as with norflurazon, a phytoene desaturase inhibitor, coloured carotenoids and chlorophyll increased with chlorpropham but decreased with norflurazon. Cultivation with chlorpropham under red LED light for 2 days did not affect the content of β-carotene, but phytoene was 2.4-fold the amount that was obtained in cultures under white LED light and the 9-cis β-carotene/all-trans β-carotene ratio increased from 1.3 to 1.8. With norflurazon, red LED light boosted the contents of both phytoene and β-carotene, 2-fold and 1.3-fold respectively compared to those under white LED light, and the ratio of 9-cis β-carotene/all-trans β-carotene reached 3.8. The results are discussed in terms of disruption by chlorpropham of synchronised control between nuclear and chloroplast events associated with carotenoid biosynthesis. Since phytoene and phytofluene are colourless carotenoids which are sought after for the development of nutricosmetics and other health/beauty products, the results also present as a new method with low toxicity for production of colourless carotenoids based on the cultivation of D. salina.

Molybdenum disulfide nanoparticles concurrently stimulated biomass and β-carotene accumulation in Dunaliella salina

Molybdenum disulfide nanoparticles (MoS2 NPs) hold tremendous properties in wide domain of applications. In this study, the impact of MoS2 NPs was investigated on algal physiological and metabolic properties and a two-stage strategy was acquired to enhance the commercial potential of Dunaliella salina. With 50 µg/L of MoS2 NPs exposure, cellular growth and biomass production were promoted by 1.47- and 1.33-fold than that in control, respectively. MoS2 NPs treated cells were subject to high light intensity for 7 days after 30 days of normal light cultivation, which showed that high light intensity gradually increased β-carotene content by 1.48-fold. Furthermore, analyses of primary metabolites showed that combinatorial approach significantly altered the biochemical composition of D. salina. Together, these findings demonstrated that MoS2 NPs at an optimum concentration combined with high light intensity could be a promising approach to concurrently enhance biomass and β-carotene production in microalgae.

Effects of different light regimes on Dunaliella salina growth and β-carotene accumulation

Outdoor cultivations of microalgae for commercial scale production usually rely on natural sunlight following ‘Sine’ regime, but most lab scale studies adopt ‘Block’ or ‘Continuous’ regimes, leading to poor prediction of outdoor cultivation results. To fill this gap on β-carotene production by Dunaliella salina, the effects of light regimes (‘Block’, ‘Sine’, ‘Continuous’) on cell growth and β-carotene accumulation in D. salina were investigated in this study. The results showed significant difference between these regimes. When receiving the same amount of illumination, ‘Sine’ mode inferred 10–55% higher biomass yield and 31–58% higher β-carotene yield than those under the ‘Block’ mode. The biomass concentration, as well as protein and total fatty acid (TFA) content, has no significant difference between different light regimes, but the starch content in the ‘Continuous’ light regime was higher than that of ‘Sine’ and ‘Block’ regime. β-carotene accumulation under ‘Continuous in summer’ and ‘Simulated diurnal irradiance in summer’ regimes were correlated with increase (C18:1, C18:2) and decrease (C16:4, C18:3) of specific fatty acids (FAs) contents, rather than TFA content. Meanwhile, it was found that β-carotene accumulation was correlated with periodical Fv/Fm fluctuations under different light regimes, and indicate that exposure to periodical strong light is more favorable for β-carotene accumulation, rather than continuous light. These results indicated the significance of using ‘Sine’ light regime since it not only provided better predictions on outdoor production, but also helpful for increase biomass and β-carotene productivities.

Carotenoids production in wild yeasts isolated from aquatic systems in Cali, Colombia

Introducción: Los carotenoides son fuente importante de actividades biológicas funcionales, tales como antioxidantes o antimicrobianas, además de tener gran impacto a nivel industrial, ya sea en cosmética o suplementación animal en acuacultura. Se han reportado varias moléculas novedosas a partir de aislamientos en Latinoamérica, principalmente en la Patagonia, Argentina. Sin embargo, no hay reportes en Colombia que evalúen la producción de carotenoides en levaduras nativas pigmentadas. Objetivo: Se evaluó la capacidad de producción de carotenoides en levaduras nativas aisladas de lagos, ríos y aguas residuales de la ciudad de Cali, Colombia. Materiales y métodos: Se caracterizaron 30 levaduras provenientes de dos colecciones. De estas se obtuvo su biomasa, rendimiento de carotenoides totales y producción de β-caroteno. Las cepas promisorias fueron identificadas secuenciando la región ITS1-5.8S-ITS2. Resultados: El mayor rendimiento en la extracción de pigmentos se obtuvo para las cepas P11A (84,36 ± 5,24 µg/g) y Rhodotorula paludigena CS13 (56,26 ± 7,08 µg/g), mientras que las concentraciones más altas de β-caroteno fueron 10,2 µg/mL (R. paludigena CS13) y 9,7 µg/mL (R. mucilaginosa/alborubescens P10A). La cinética de crecimiento y producción de pigmentos durante cinco días fue óptima para la cepa P11A, ya que hubo un aumento en el rendimiento de carotenoides totales 10 veces mayor (48 h: 109,62 µg/g, 120 h: 1403,10 µg/g). Conclusiones: En este estudio se encontró que levaduras aisladas de sistemas acuáticos son promisorias para la producción de pigmentos carotenoides (incluyendo β-caroteno), siendo su extracción y caracterización viable para futuros estudios biotecnológicos.

Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of \u03b2-carotene in Saccharomyces cerevisiae

BackgroundProduct toxicity is one of the bottlenecks for microbial production of biofuels, and transporter-mediated biofuel secretion offers a promising strategy to solve this problem. As a robust microbial host for industrial-scale production of biofuels, Saccharomyces cerevisiae contains a powerful transport system to export a wide range of toxic compounds to sustain survival. The aim of this study is to improve the secretion and production of the hydrophobic product (β-carotene) by harnessing endogenous ABC transporters combined with physiological engineering in S. cerevisiae.ResultsSubstrate inducibility is a prominent characteristic of most endogenous transporters. Through comparative proteomic analysis and transcriptional confirmation, we identified five potential ABC transporters (Pdr5p, Pdr10p, Snq2p, Yor1p, and Yol075cp) for β-carotene efflux. The accumulation of β-carotene also affects cell physiology in various aspects, including energy metabolism, mitochondrial translation, lipid metabolism, ergosterol biosynthetic process, and cell wall synthesis. Here, we adopted an inducible GAL promoter to overexpress candidate transporters and enhanced the secretion and intracellular production of β-carotene, in which Snq2p showed the best performance (a 4.04-fold and a 1.33-fold increase compared with its parental strain YBX-01, respectively). To further promote efflux capacity, two strategies of increasing ATP supply and improving membrane fluidity were following adopted. A 5.80-fold increase of β-carotene secretion and a 1.71-fold increase of the intracellular β-carotene production were consequently achieved in the engineered strain YBX-20 compared with the parental strain YBX-01.ConclusionsOverall, our results showcase that engineering endogenous plasma membrane ABC transporters is a promising approach for hydrophobic product efflux in S. cerevisiae. We also highlight the importance of improving cell physiology to enhance the efficiency of ABC transporters, especially energy status and cell membrane properties.

Growth factors arrangement enhances Thermosynechococcus sp. CL-1 carotenoid productivity during CO2 fixation

CO2 bio-fixation by photosynthetic microorganisms not only offer high efficiency on its process, but also produce valuable byproducts. Carotenoids are one of the interesting byproducts of the photosynthetic microorganism cultivation due to its wide application in industries. Isolated cyanobacterium, Thermosynechococcus sp. CL-1 (TCL-1), was assessed for its theoretical feasibility of performance on biomass productivity, carotenoid (zeaxanthin, β-carotene) productivity and CO2 fixation rate. The cultivation carried out under various growth factors including light intensity, concentration of nitrogen supply and salinity.Cultivation with the addition of 5.8 mM nitrogen supply under light intensity of 1000 μE/m2/s and 0.14 M salinity gained the best results on biomass productivity (90.3 mg/L/h) and CO2 fixation rate (129.1 ± 5.5 mg/L/h) at the 8th hour of cultivation. This results related with the highest zeaxanthin productivity which reached 0.074 ± 0.004 mg/L/h and β-carotene productivity of 0.39 ± 0.03 mg/L/h. The results indicated that TCL-1 is the potential source to efficiently produce carotenoid compounds.

Production of β-carotene with Dunaliella salina CCAP19/18 at physically simulated outdoor conditions.

Batch growth and β‐carotene production of Dunaliella salina CCAP19/18 was investigated in flat‐plate gas‐lift photobioreactors with a light path of 2 cm, operated in physically simulated outdoor conditions. Dunaliella salina CCAP19/18 showed robust growth with respect to pH 8.0‐9.0 and 15–35°C at increasing salinity, simulating the evaporation of open photobioreactors. The highest β‐carotene concentration of 25 mg L‐1 (3 mg gCDW −1) was observed in batch processes at pH 8.5, 15–30°C and increasing salinity up to 110 g L‐1, simulating a typical Mediterranean summer climate. Intracellular β‐carotene accumulation of D. salina CCAP19/18 was shown to be independent of light availability, although nutrient limitation (K2HPO4, MgSO4, and/or ammonium ferric citrate) seems to enable stable β‐carotene content in the algal cells despite increasing cell densities in the photobioreactor. Fully controlled, lab‐scale photobioreactors simulating typical climate conditions of any region of interest are valuable tools for enabling a realistic characterization of microalgae on a laboratory scale, for production processes projected in open photobioreactor systems (e.g. thin‐layer cascade photobioreactors).

Metabolic pathway optimization through fusion with self-assembling amphipathic peptides

Fusion with self-assembling amphipathic peptides (SAPs) could improve enzyme stability and activity, as well as enhancing protein expression. In this study, we proposed a metabolic regulation strategy based on an iterative SAP fusion with key enzymes in the pathway. First, an SAP, S1nv10 (ANANARARANANARAR), was separately fused to the N-terminus of metabolite enzymes. To achieve diversity in the enzyme activities, a library of the fused SAPs that varied in hydrophobicity, length, and net charge was generated by a multiprimer PCR procedure and the DATEL-assembly method. After simultaneous modification of three enzymes (CrtE, CrtY, and CrtI) using the SAP library, an Escherichia coli strain with a 3.91-fold increase in β-carotene production was isolated. As indicated by western blotting and catalytic property analysis, SAP fusion induced increases in both enzyme expression (CrtY and CrtE) and enzymatic activities (CrtY). The effectiveness of the strategy was further confirmed by the metabolic regulation of eriodictyol and (2S)-naringenin, the yields of which were increased by 111.3 % and 186.3 % in E. coli, respectively. These results indicate that fusing SAPs to the N-terminus of enzymes in metabolic flux is an efficient strategy for metabolic engineering.

Enhanced Production of Astaxanthin without Decrease of DHA Content in Aurantiochytrium limacinum by Overexpressing Multifunctional Carotenoid Synthase Gene.

Aurantiochytrium limacinum produces both docosahexaenoic acid (DHA) and astaxanthin, respectively. Organisms that produce these industrially important materials more efficiently than microalgae are currently needed. In this study, we overexpressed a putative homolog of CarS, which is involved in synthesizing the astaxanthin precursor, β-carotene, in A. limacinum to increase carotenoid synthesis with the goal of obtaining strains that produce large amounts of both DHA and carotenoids. AlCarS transformants #1 and #18 produced significantly increased amounts of astaxanthin as assessed according to culture (up to 5.8-fold) and optical density (up to 9.3-fold). The improved astaxanthin production of these strains did not affect their DHA productivity. Additionally, their CarS expression levels were higher than those of the wild-type strain, suggesting that CarS overexpression enhanced β-carotene production, which in turn improved astaxanthin productivity. Although cell yields were slightly decreased, these features will be valuable in health food, medical care, and animal feed fields.