Lycopene Yield Research Articles

Enzyme-aided extraction of lycopene from high-pigment tomato cultivars by supercritical carbon dioxide

This work reports a novel enzyme-assisted process for lycopene concentration into a freeze-dried tomato matrix and describes the results of laboratory scale lycopene supercritical CO2 (SC-CO2) extractions carried out with untreated (control) and enzyme-digested matrices. The combined use of food-grade commercial plant cell-wall glycosidases (Celluclast/Novozyme plus Viscozyme) allows to increase lycopene (∼153%) and lipid (∼137%) concentration in the matrix and rises substrate load onto the extraction vessel (∼46%) compared to the control. The addition of an oleaginous co-matrix (hazelnut seeds) to the tomato matrix (1:1 by weight) increases CO2 diffusion through the highly dense enzyme-treated matrix bed and provides lipids that are co-extracted increasing lycopene yield. Under the same operative conditions (50MPa, 86°C, 4mLmin−1 SC-CO2 flow) extraction yield from control and Celluclast/Novozyme+Viscozyme-treated tomato matrix/co-matrix mixtures was similar, exceeding 75% after 4.5h of extraction. However, the total extracted lycopene was ∼3 times higher in enzyme-treated matrix than control.

Production of lycopene by metabolically-engineered Escherichia coli.

Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5mg lycopene/l (6.5mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76% increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32%. The optimal strain LYC010 produced 3.52g lycopene/l (50.6mg/g DCW) in fed-batch fermentation.

Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10-45% increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174% in shake-flask and 81% in bioreactor fermentations with a maximum yield of 454 mg l(-1).

Combining Genotype Improvement and Statistical Media Optimization for Isoprenoid Production in E. coli

Isoprenoids are a large and diverse class of compounds that includes many high value natural products and are thus in great demand. To meet the increasing demand for isoprenoid compounds, metabolic engineering of microbes has been used to produce isoprenoids in an economical and sustainable manner. To achieve high isoprenoid yields using this technology, the availability of metabolic precursors feeding the deoxyxylulose phosphate (DXP) pathway, responsible for isoprenoid biosynthesis, has to be optimized. In this study, phosphoenolpyruvate, a vital DXP pathway precursor, was enriched by deleting the genes encoding the carbohydrate phosphotransferase system (PTS) in E. coli. Production of lycopene (a C40 isoprenoid) was maximized by optimizing growth medium and culture conditions. In optimized conditions, the lycopene yield from PTS mutant was seven fold higher than that obtained from the wild type strain. This resulted in the highest reported specific yield of lycopene produced from the DXP pathway in E. coli to date (20,000 µg/g dry cell weight). Both the copy number of the plasmid encoding the lycopene biosynthetic genes and the expression were found to be increased in the optimized media. Deletion of PTS together with a similar optimization strategy was also successful in enhancing the production of amorpha-1,4-diene, a distinct C15 isoprenoid, suggesting that the approaches developed herein can be generally applied to optimize production of other isoprenoids.

Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase

Genetic manipulation was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli. Knockout of the central carbon metabolic gene zwf (glucose-6-phosphate dehydrogenase) resulted in the enhancement of lycopene production (above 130 % relative to control). The amplification and overexpression of rate-limiting steps encoded by idi (isopentenyl diphosphate isomerase), dxs (1-deoxyxylulose-5-phosphate synthase) and ispDF (4-diphosphocytidyl-2C-methyl-D-erythritol synthase and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase) genes improved lycopene synthesis from 0.89 to 5.39 mg g(-1) DCW. The combination of central metabolic genes knockout with the amplification of MEP pathway genes yielded best amounts of lycopene (6.85-7.55 mg g(-1) DCW). Transcript profiling revealed that idi and dxs were up-regulated in the zwf knock-out strain, providing a plausible explanation for the increase in lycopene yield observed in this strain. An increase in precursor availability might also have contributed to the improved lycopene production.

Enhanced Lycopene Content in Blakeslea trispora by Effective Mutation-Screening Method

The zygomycete fungus Blakeslea trispora is usually used as a natural source of lycopene and β-carotene. In this study, the B. trispora (-) strain, a major mating type for lycopene production, was treated with N(+) ion implantation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid (CTA). After several rounds of screening, four mutants with higher yield of lycopene and biomass were isolated. Among these mutants, I5 obtained with N(+) ion implantation showed a maximum lycopene yield (28.8 mg/g), which was 64 % higher than the parent strain (17.5 mg/g) in the production of lycopene. The results indicated that N(+) ion implantation is more suitable for B. trispora (-) than NTG treatment, and the addition of lovastatin promoted the generation of positive mutant and CTA amplified the color differences between colonies.

Effects of pre-treatments on the yield and carotenoid content of Gac oil using supercritical carbon dioxide extraction

The effects of air-drying temperature, particle size and enzymatic pre-treatment on the oil yield and content of carotenoids in the resultant Gac oil using supercritical CO2 extraction method were investigated. It was found that the highest oil yield was obtained when using the enzyme concentration at 0.1% (w/w) for pretreatment before air-drying. Furthermore, the content of carotenoids was also enhanced by suitable drying temperature and the particle size. High oil yield (34% g/g) and high content of β-carotene (83mg/100mL oil) and lycopene (508mg/100mL oil) were obtained using the drying temperature of 50°C and particle size of 0.45mm as pre-treatments prior to SC-CO2 extraction at pressure of 200bar and extraction temperature of 50°C. Results also indicated that the most suitable extraction time was 120min. It was concluded that Gac oil containing high amount of carotenoids could be extracted using the chosen air-drying temperature, particle size, enzymatic pre-treatment and extraction time.

Ethanol production by repeated batch fermentation of sweet sorghum juice utilizing thermotolerant yeast Kluyveromyces marxianus DBKKUY-103

Lycopene, a tetraterpenoid containing 13 double bonds, has important pharmaceutical and industrial application values. However, unstable supply of raw for extracted from natural host and residue of chemical reagents in chemically synthesized products have limited the quality of lycopene. Engineering microorganism is thus regarded as an alternative and attractive route for lycopene production. To improve the ability of engineered yeast to synthesize lycopene, here we have optimized the lycopene biosynthesis pathway and chassis metabolism. The results have verified that S. cerevisiae CEN.PK2-1C was more suitable for lycopene production because of its strong ability of synthesizing precursors of terpenoids. Compared to use inducible promoter, the use of constitutive promoter controlling pathway expression was more efficient for balancing lycopene synthesis and chassis metabolism. GGPP was identified the rate-controlling metabolite. Through expanding GGPP pool and MVA pathway, lycopene production was thus significantly increased. By citric acid fed-batch fermentation, the yield of lycopene finally reached 115.64 mg/L, 2689-folds of initial engineered yeast. These results indicated that co-optimization of heterologous pathway and chassis metabolism is a good strategy for enforcing microbial overproduction of natural products.

Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains

NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2-haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6-phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose

While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched 13C-lycopene for human bioavailability and metabolism studies. To enhance the 13C-enrichment and yields of labelled lycopene from the hp-1 tomato cell line, cultures were first grown in 13C-glucose media for three serial batches and produced increasing proportions of uniformly labelled lycopene (14.3±1.2%, 39.6±0.5%, and 48.9±1.5%) with consistent yields (from 5.8 to 9mg/L). An optimised 9-day-long 13C-loading and 18-day-long labelling strategy developed based on glucose utilisation and lycopene yields, yielded 13C-lycopene with 93% 13C isotopic purity, and 55% of isotopomers were uniformly labelled. Furthermore, an optimised acetone and hexane extraction led to a fourfold increase in lycopene recovery from cultures compared to a standard extraction.

Ultrasound assisted extraction of lycopene from tomato processing wastes.

Tomato paste processing wastes, an important by-product of the paste industry, is rich in potentially health-promoting compounds such as lycopene. In this study, extraction yield of lycopene from tomato paste processing wastes by application of ultrasound assisted extraction (UAE) was compared with conventional organic solvent extraction (COSE) method. BHT (butylated hydroxytoluene) 0.05% (w/v) added hexane:acetone:ethanol (2:1:1) mixture was used as solvent. Three different solvent solid ratios; 50:1, 35:1 and 20:1, (v/w) were used in both COSE and UAE. COSE experiments were performed at 20°C, 40°C and 60°C for 10, 20, 30 and 40min. 50, 65 and 90W of ultrasonic power were applied in UAE for 1, 2, 5, 10, 15, 20 and 30min. Lycopene contents of the samples were determined by spectrophotometric method. The effects of different factors, including the temperature, solvent solid ratio and ultrasonic power on lycopene yield were investigated. It was determined that the most efficient application for COSE was extracting samples by 50:1 solvent solid ratio at 60°C for 40min run, for UAE, 35:1 (v/w) solvent solid ratio, 90W ultrasonic power for 30min run. It was showed that UAE of lycopene requires less time, lower temperature and lower solvent than COSE.

Novel modified ultrasonication technique for the extraction of lycopene from tomatoes

Lycopene extraction was carried out via the ultrasonic assisted extraction (UAE) with response surface methodology (RSM). Sonication enhanced the efficiency of relative lycopene yield (enhancement of 26% extraction yield of lycopene in 6 replications at 40.0 min, 40.0 °C and 70.0% v/w in the presence of ultrasound), lowered the extraction temperature and shortened the total extraction time. The extraction was applied with the addition of oxygen-free nitrogen flow and change of water route during water bath sonication. The highest relative yield of lycopene obtained was 100% at 45.0 °C with total extraction time of 50.0 min (30:10:10) and ratio of solvent to freeze-dried tomato sample (v/w) of 80.0:1. Optimisation of the lycopene extraction had been performed, giving the average relative lycopene yield of 99% at 45.6 min, 47.6 °C and ratio of solvent to freeze-dried tomato sample (v/w) of 74.4:1. From the optimised model, the average yield of all- trans lycopene obtained was 5.11 ± 0.27 mg/g dry weight. The all- trans lycopene obtained from the high-performance liquid chromatography (HPLC) chromatograms was 96.81 ± 0.81% with 3.19 ± 0.81% of cis-lycopenes. The purity of total-lycopene obtained was 98.27 ± 0.52% with β-carotene constituted 1.73 ± 0.52% of the extract. The current improved, UAE of lycopene from tomatoes with the aid of RSM also enhanced the extraction yield of trans-lycopene by 75.93% compared to optimised conventional method of extraction. Hence, the current, improved UAE of lycopene promotes the extraction yield of lycopene and at the same time, minimises the degradation and isomerisation of lycopene.

Effect of aqueous extract of Sargassum johnstonii Setchell & Gardner on growth, yield and quality of Lycopersicon esculentum Mill.

Experiments were conducted on tomato to study the potential of brown alga Sargassum johnstonii as a biofertilizer. Seaweed extract was applied as a foliar spray, soil drench, and soil drench + foliar spray to assess its effect on plant growth, yield, and concentration of lycopene and vitamin C. The main objective of the study was to enhance the biochemical constituents with neutraceutical and antioxidant values in tomato fruit. Different concentrations (0.1%, 0.4%, 0.8%, 2%, 6%, 8%, and 10%; v/v) of seaweed extract were used and growth was observed over a period of 7 months. A total of 14 sprays/drenches were applied at 15-day intervals during the entire vegetative and reproductive phase. A statistically significant increase in vegetative growth (plant height, shoot length, root length, and number of branches), reproductive parameters (flower number, fruit number, and fresh weight), and biochemical constituents (photosynthetic pigments, proteins, total soluble sugars, reducing sugars, starch, phenols, lycopene, and vitamin C) was recorded following all three methods of treatment at higher concentrations of seaweed extract. The study also reports auxin- and cytokinin-like activity, and the presence of macro- (Ca, Mg, Na, and K) and micronutrients (Fe, Cu, Zn, and Mn), in seaweed extract of S. johnstonii, which makes it a potential biofertilizer.

Stabilized gene duplication enables long-term selection-free heterologous pathway expression

Engineering robust microbes for the biotech industry typically requires high-level, genetically stable expression of heterologous genes and pathways. Although plasmids have been used for this task, fundamental issues concerning their genetic stability have not been adequately addressed. Here we describe chemically inducible chromosomal evolution (CIChE), a plasmid-free, high gene copy expression system for engineering Escherichia coli. CIChE uses E. coli recA homologous recombination to evolve a chromosome with approximately 40 consecutive copies of a recombinant pathway. Pathway copy number is stabilized by recA knockout, and the resulting engineered strain requires no selection markers and is unaffected by plasmid instabilities. Comparison of CIChE-engineered strains with equivalent plasmids revealed that CIChE improved genetic stability approximately tenfold and growth phase-specific productivity approximately fourfold for a strain producing the high metabolic burden-biopolymer poly-3-hydroxybutyrate. We also increased the yield of the nutraceutical lycopene by 60%. CIChE should be applicable in many organisms, as it only requires having targeted genomic integration methods and a recA homolog.

High-level production of lycopene in metabolically engineered E. coli

Recombinant lycopene was generated by utilizing metabolically engineered Escherichia coli with yields being dependent upon inocula state. Yields were especially low in the case of cultures harboring high-copy plasmids that were established with inocula at the stationary growth phase. On the other hand, cultures derived using low-copy plasmid, however, yielded high amounts of lycopene irrespective of inocula state. Nevertheless, it showed still an inocula dependence pattern in lycopene productivity (mg/l/h). To further increase lycopene productivity, we applied a temperature-shift culture technique (37 → 25 °C). Using this method, we effectively enhanced lycopene productivity without any problematic phenomena. As a result, we were able to increase lycopene yield by approximately 20% compared to previous culture methods. In the present study, we were able to reach a final lycopene yield up to 260 mg/l for 60 h, which corresponds to the highest titer to date for the production of lycopene in E. coli.

THE EFFECT OF WATER AND POTASSIUM SUPPLEMENT ON YIELD AND LYCOPENE CONTENT OF PROCESSING TOMATO

THE EFFECT OF WATER AND POTASSIUM SUPPLEMENT ON YIELD AND LYCOPENE CONTENT OF PROCESSING TOMATO

Effects of modifiers on the profile of lycopene extracted from tomato skins by supercritical CO 2

The effects of the addition of non-toxic modifiers (ethanol, water, and canola oil) in supercritical carbon dioxide (SC-CO 2) extractions from tomato skins were investigated. The extraction efficiency was improved by the addition of any of the three modifiers. Various temperatures (45 °C, 60 °C, and 75 °C) and pressures (25, 30, and 35 MPa) for the modified extraction processes were also compared. The highest yield was achieved at the highest temperature (75 °C) and highest pressure (35 MPa). For the three single modifiers, yields increased with increasing amounts (from 5% to 10%) of the modifiers. The highest lycopene yields were obtained with 15% olive oil at 45 °C and 10% olive oil at 75 °C. For mixtures of binary and ternary modifiers, the mixture of ethanol (10%) and olive oil (10%) achieved the highest extraction efficiency (36.6% at 45 °C, and 56.8% at 75 °C). It might be caused by the combined synergetic influences of enhanced adsorption on the polar sites of the matrix by ethanol and the increased solubility of lycopene by olive oil. Cluster analysis was applied to build the relationship between the lycopene yields and co-solvent effects. The addition of olive oil also slightly increased the proportion of cis-isomers in the lycopene extract.

Effects of supercritical fluid extraction parameters on lycopene yield and antioxidant activity

The effects of various parameters of supercritical carbon dioxide (SC-CO 2) fluid extractions of tomato skins on the extraction yields and antioxidant activities of lycopene-rich extracts were investigated. A Box–Behnken design was applied to study the effects of three independent variables (temperature ranging from 40 to 100 °C, pressure ranging from 20 to 40 MPa, and flow rate ranging from 1.0 to 2.0 mL/min) on lycopene yield. The model showed good agreement with the experimental results, by the coefficient of determination ( r 2 = 0.9834). Temperature, pressure, and the quadratic term for the temperature of SC-CO 2 extraction were large significantly positive factors affecting lycopene yield ( P < 0.05). The maximum total lycopene content of 31.25 μg/g of raw tomato was extracted at the highest temperature of 100 °C, 40 MPa and 1.5 mL/min. TEAC assay was applied to assess the antioxidant activity of lycopene-rich extracts from SC-CO 2 fluid extraction. The effects of SC-CO 2 fluid extraction parameters on the antioxidant activities of the extracts differed with the yield. For each unit of lycopene extract, the antioxidant activity level was constant below 70 °C, but then gradually decreased above 70 °C due to isomerization occurring as a result of the higher temperature. The ratio of all- trans-lycopene to the cis-isomers changed from 1.70 to 1.32 when the operating temperature was adjusted from 40 to 100 °C, indicating an increased bioavailability due to the generation of the cis-isomers. No significant effects of pressure or flow rate of SC-CO 2 fluid extraction on the antioxidant activity were observed.

Optimization of Lycopene Extraction from Tomato Cell Suspension Culture by Response Surface Methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

Lycopene Production Using Blakeslea trispora in the Presence of 2-Methyl Imidazole: Yield, Selectivity, and Safety Aspects

The potential role of 2-methyl imidazole in improving lycopene production by Blakeslea trispora with regards to yield, selectivity, and safety aspects was investigated in batch culture. Optimization of the bioprocess conditions in terms of (a) (+) and (-) strain ratio in the inoculum, (b) initial crude soybean oil (CSO) addition level, and (c) the amount of 2-methyl imidazole was based on response surface methodology to achieve maximum lycopene production. The dependence of growth kinetics, lycopene yield, and selectivity of the bioprocess on the above factors was clear. 2-Methyl imidazole at 50 mg/L was found equally active in terms of lycopene cyclase inhibition with that at 200 or 100 mg/L; in all cases, lycopene accounted for 94% of the total carotenoids. The highest yield was observed at a 50 mg/L level of addition (24 mg/g of biomass dry weight,) in a substrate supplemented with CSO (48 g/L of culture medium) and inoculated with 1(+)/7(-) strain ratio.