JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Efficient free fatty acid production from woody biomass hydrolysate using metabolically engineered Escherichia coli.
Bioresour. Technol.
PUBLISHED: 05-05-2014
Show Abstract
Hide Abstract
Four engineered Escherichia coli strains, ML103(pXZ18), ML103(pXZ18Z), ML190(pXZ18) and ML190(pXZ18Z), were constructed to investigate free fatty acid production using hydrolysate as carbon source. These strains exhibited efficient fatty acid production when xylose was used as the sole carbon source. For mixed sugars, ML103 based strains utilized glucose and xylose sequentially under the carbon catabolite repression (CCR) regulation, while ML190 based strains, with ptsG mutation, used glucose and xylose simultaneously. The total free fatty acid concentration and yield of the strain ML190(pXZ18Z) based on the mixed sugar reached 3.64 g/L and 24.88%, respectively. Furthermore, when hydrolysate from a commercial plant was used as the carbon source, the strain ML190(pXZ18Z) can produce 3.79 g/L FFAs with a high yield of 21.42%.
Related JoVE Video
Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol.
Metab. Eng.
PUBLISHED: 04-21-2014
Show Abstract
Hide Abstract
Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl-acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.
Related JoVE Video
Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli.
Biotechnol. Bioeng.
PUBLISHED: 03-13-2014
Show Abstract
Hide Abstract
Free fatty acids (FFAs) can be used as precursors for the production of biofuels or chemicals. Different composition of FFAs will be useful for further modification of the biofuel/biochemical quality. Microbial biosynthesis of even chain FFAs can be achieved by introducing an acyl-acyl carrier protein thioesterase gene into E. coli. In this study, odd straight medium chain FFAs production was investigated by using metabolic engineered E. coli carrying acyl-ACP thioesterase (TE, Ricinus communis), propionyl-CoA synthase (Salmonella enterica), and ?-ketoacyl-acyl carrier protein synthase III (four different sources) with supplement of extracellular propionate. By using these metabolically engineered E. coli, significant quantity of C13 and C15 odd straight-chain FFAs could be produced from glucose and propionate. The highest concentration of total odd straight chain FFAs attained was 1205?mg/L by the strain HWK201 (pXZ18, pBHE2), and 85% of the odd straight chain FFAs was C15. However, the highest percentage of odd straight chain FFAs was achieved by the strain HWK201 (pXZ18, pBHE3) of 83.2% at 48?h. This strategy was also applied successfully in strains carrying different TE, such as the medium length acyl-ACP thioesterase gene from Umbellularia californica. C11 and C13 became the major odd straight-chain FFAs.
Related JoVE Video
Engineering Escherichia coli for odd straight medium chain free fatty acid production.
Appl. Microbiol. Biotechnol.
PUBLISHED: 02-20-2014
Show Abstract
Hide Abstract
Microbial biosynthesis of free fatty acids (FFAs) can be achieved by introducing an acyl-acyl carrier protein thioesterase gene into Escherichia coli. The engineered E. coli usually produced even chain FFAs. In this study, propionyl-CoA synthetase (prpE) from Salmonella enterica was overexpressed in two efficient even chain FFAs producers, ML103 (pXZM12) carrying the acyl-ACP thioesterase gene from Umbellularia californica and ML103 (pXZ18) carrying the acyl-ACP thioesterase gene from Ricinus communis combined with supplement of extracellular propionate. With these metabolically engineered E. coli, the odd straight chain FFAs, undecanoic acid (C11:0), tridecanoic acid (C13:0), and pentadecanoic acid (C15:0) were produced from glucose and propionate. The highest total odd straight chain FFAs produced by ML103 (pXZM12, pBAD-prpE) reached 276 mg/l with a ratio of 23.43 % of the total FFAs. In ML103 (pXZ18, pBAD-prpE), the highest total odd straight chain FFAs accumulated to 297 mg/l, and the ratio reached 17.68 % of the total FFAs. Due to the different substrate specificity of the acyl-ACP thioesterases, the major odd straight chain FFA components of ML103 (pXZM12, pBAD-prpE) were undecanoic acid and tridecanoic acid, while the ML103 (pXZ18, pBAD-prpE) preferred pentadecanoic acid.
Related JoVE Video
Improvement of NADPH bioavailability in Escherichia coli by replacing NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with NADP (+)-dependent GapB from Bacillus subtilis and addition of NAD kinase.
J. Ind. Microbiol. Biotechnol.
PUBLISHED: 04-24-2013
Show Abstract
Hide Abstract
Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichia coli was replaced with a NADP(+)-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP(+) availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD(+)-dependent GapA activity with NADP(+)-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds.
Related JoVE Video
Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD(+)-dependent formate dehydrogenase.
Metab. Eng.
PUBLISHED: 04-22-2013
Show Abstract
Hide Abstract
Succinic acid is a specialty chemical having numerous applications in industrial, pharmaceutical and food uses. One of the major challenges in the succinate fermentation process is eliminating the formation of byproducts. In this study, we describe eliminating byproduct formate and improving succinate productivity by reengineering a high succinate producing E. coli strain SBS550MG-Cms243(pHL413Km). The NAD(+)-dependent formate dehydrogenase gene (fdh1) of Candida boidinii was coexpressed with Lactococcus lactis pyruvate carboxylase (pycA) under the control of Ptrc and PpycA promoters in plasmid pHL413KF1. The newly introduced fdh1 converts 1mol of formate into 1mol of NADH and CO2. The reengineered strain SBS550MG-Cms243(pHL413KF1) retains the reducing power of formate through an increase in NADH availability. In anaerobic shake flask fermentations, the parent strain SBS550MG-Cms243(pHL413Km) consumed 99.86mM glucose and produced 172.38mM succinate, 16.16mM formate and 4.42mM acetate. The FDH bearing strain, SBS550MG-Cms243(pHL413KF1) consumed 98.43mM glucose and produced 171.80mM succinate, 1mM formate and 5.78mM acetate. Furthermore, external formate supplementation to SBS550MG(pHL413KF1) fermentations resulted in about 6% increase in succinate yields as compared to SBS550MG(pHL413Km). In an anaerobic fed-batch bioreactor process, the average glucose consumption rate, succinate productivity, and byproduct formate concentration of SBS550MG(pHL413Km) was 1.40g/L/h, 1g/L/h, and 17mM, respectively. Whereas, the average glucose consumption rate, succinate productivity and byproduct formate concentration of SBS550MG(pHL413KF1) was 2g/L/h, 2g/L/h, 0-3mM respectively. A high cell density culture of SBS550MG(pHL413KF1) showed further improvement in succinate productivity with a higher glucose consumption rate. Reduced levels of byproduct formate in succinate fermentation broth would provide an opportunity for reducing the cost associated with downstream processing, purification, and waste disposal.
Related JoVE Video
Metabolic engineering and transhydrogenase effects on NADPH availability in escherichia coli.
Biotechnol. Prog.
PUBLISHED: 04-08-2013
Show Abstract
Hide Abstract
The synthesis of several industrially useful compounds are cofactor-dependent, requiring reducing equivalents like NADPH in enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, NADPH is costly and has limited intracellular availability. This study focuses on the study of the effect of the two transhydrogenase enzymes of Escherichia coli, PntAB and UdhA (SthA) on reducing equivalents-dependent biosynthesis. The production of (S)-2-chloropropionate from 2-chloroacrylate is used as a model system for monitoring NADPH availability because 2-haloacrylate reductase, the enzyme catalyzing the one-step conversion to (S)-2-chloropropionate in the synthesis pathway, requires NADPH as a cofactor. Results suggest that the presence of UdhA increases product yield and NADPH availability while the presence of PntAB has the opposite effect. A maximum product yield of 1.4 mol product/mol glucose was achieved aerobically in a pnt-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
Related JoVE Video
Metabolic engineering and transhydrogenase effects on NADPH availability in escherichia coli.
Biotechnol. Prog.
PUBLISHED: 04-08-2013
Show Abstract
Hide Abstract
The synthesis of several industrially useful compounds are cofactor-dependent, requiring reducing equivalents like NADPH in enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, NADPH is costly and has limited intracellular availability. This study focuses on the study of the effect of the two transhydrogenase enzymes of Escherichia coli, PntAB and UdhA (SthA) on reducing equivalents-dependent biosynthesis. The production of (S)-2-chloropropionate from 2-chloroacrylate is used as a model system for monitoring NADPH availability because 2-haloacrylate reductase, the enzyme catalyzing the one-step conversion to (S)-2-chloropropionate in the synthesis pathway, requires NADPH as a cofactor. Results suggest that the presence of UdhA increases product yield and NADPH availability while the presence of PntAB has the opposite effect. A maximum product yield of 1.4 mole product/mole glucose was achieved aerobically in a pnt-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
Related JoVE Video
Cofactor engineering for advancing chemical biotechnology.
Curr. Opin. Biotechnol.
PUBLISHED: 03-21-2013
Show Abstract
Hide Abstract
Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.
Related JoVE Video
Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains.
Appl. Microbiol. Biotechnol.
PUBLISHED: 03-13-2013
Show Abstract
Hide Abstract
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.
Related JoVE Video
Screening 64 cultivars Catharanthus roseus for the production of vindoline, catharanthine, and serpentine.
Biotechnol. Prog.
PUBLISHED: 06-14-2011
Show Abstract
Hide Abstract
The leaves of Catharanthus roseus (L.) G. Don. are a valuable source of the terpenoid indole alkaloid (TIA) anticancer drugs, vinblastine and vincristine. In particular, the precursor molecules vindoline and catharanthine are harvested from leaves and used for the semisynthetic production of vinblastine and vincristine. Because of this application, catharanthine and vindoline can be used to screen for high-yielding TIA cultivars. In this study, we compared the TIA concentrations in the leaves of 64 different cultivars of C. roseus in the soil experiments. The highest concentration of serpentine was found in Cooler Rose Hot (461±46 ?g/g DW). Concentrations of vindoline (2082±113 ?g/g DW) and catharanthine (2903±384 ?g/g DW) were highest in Pacifica Peach. To eliminate the abiotic and biotic effects of the soils on the plant growth, sterile agar experiments were performed to investigate the TIA concentrations and mRNA transcript levels of selected TIA pathway genes. Six cultivars were investigated (two each of the high level, mid level, and low level producers of TIAs).
Related JoVE Video
Manipulating respiratory levels in Escherichia coli for aerobic formation of reduced chemical products.
Metab. Eng.
PUBLISHED: 06-08-2011
Show Abstract
Hide Abstract
Optimizing the productivity of bioengineered strains requires balancing ATP generation and carbon atom conservation through fine-tuning cell respiration and metabolism. Traditional approaches manipulate cell respiration by altering air feeding, which are technically difficult especially in large bioreactors. An approach based on genetic regulation may better serve this purpose. With excess oxygen supply to the culture, we efficiently manipulated Escherichia coli cell respiration by adding different amount of coenzyme Q1 to strains lacking the ubiCA genes, which encode two critical enzymes for ubiquinone synthesis. As a proof-of-concept, the metabolic effect of the ubiCA gene knockout and coenzyme Q1 supplementation were characterized, and the metabolic profiles of the experimental strains showed clear correlations with coenzyme Q1 concentrations. Further proof-of-principle experiments were performed to illustrate that the approach can be used to optimize cell respiration for the production of chemicals of interest such as ethanol. This study showed that controlled respiration through genetic manipulation can be exploited to allow much larger operating windows for reduced product formation even under fully aerobic conditions.
Related JoVE Video
Succinate production in Escherichia coli.
Biotechnol J
PUBLISHED: 06-02-2011
Show Abstract
Hide Abstract
Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.
Related JoVE Video
Improving fatty acid production in Escherichia coli through the overexpression of malonyl coA-acyl carrier protein transacylase.
Biotechnol. Prog.
PUBLISHED: 04-19-2011
Show Abstract
Hide Abstract
The microbial biosynthesis of free fatty acid, which can be used as precursors for the production of fuels or chemicals from renewable carbon sources, has attracted significant attention in recent years. Free fatty acids can be produced by introducing an acyl-carrier protein (ACP) thioesterase (TE) gene into Escherichia coli. The first committed step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl-CoA by an adenosine triphosphate (ATP)-dependent acetyl-CoA carboxylase followed by the conversion of malonyl-CoA to malonyl-ACP through the enzyme malonyl CoA-acyl carrier protein transacylase (MCT; FabD). The E. coli fabD gene encoding MCT has been cloned and studied. However, the effect of FabD overexpression in a fatty acid overproducing strain has not been examined. In this study, we examined the effect of FabD overexpression in a fatty acid overproducing strain carrying an acyl-ACP TE. Specifically, the effect of overexpressing a fabD gene from four different organisms on fatty acid production was compared. The strains carrying a fabD gene from E. coli, Streptomyces avermitilis MA-4680, or Streptomyces coelicolor A3(2) improved the free fatty acid production; these three strains produced more free fatty acids, about 11% more, than the control strain. The strain carrying a fabD gene from Clostridium acetobutylicum ATCC 824, however, produced similar quantities of free fatty acids as the control strain. In addition, the three FabD overexpressed strains also have higher fatty acid/glucose yields. The results suggested that FabD overexpression can be used to improve free fatty acid production by increasing the malonyl-ACP availability.
Related JoVE Video
Succinate production from sucrose by metabolic engineered Escherichia coli strains under aerobic conditions.
Biotechnol. Prog.
PUBLISHED: 03-27-2011
Show Abstract
Hide Abstract
Two metabolically engineered E. coli strains HL2765k and HL27659k, while capable of producing succinate from glucose with high yields, are not able to grow and produce succinate on sucrose. Consequently, the pUR400 plasmid containing scrK, Y, A, B, and R genes was introduced into HL2765k and HL27659k, respectively. Shake flask culture studies showed that the resulting strains can utilize sucrose; the strain HL2765k pUR400 and HL27659k pUR400 can produce succinate aerobically with a molar yield of 0.78 ± 0.02 mol/mol and 1.35 ± 0.13 mol/mol, respectively. On introduction of the plasmid pHL413, which encodes the heterologous pyruvate carboxylase (PYC) from Lactococcus lactis, the molar succinate yield increased to 1.60 ± 0.01 mol of succinate per mole of sucrose by the HL2765k pUR400 pHL413 strain and to 1.84 ± 0.10 by the HL27659k pUR400 pHL413 strain. In aerobic batch bioreactor studies, the succinate production rate was faster, and succinate production reached 101.83 mM with a yield of 1.90 when dissolved oxygen (DO) was controlled at 40 ± 7%. In addition, the results showed that DO had an important effect on succinate production by influencing PYC activity. This work demonstrates the possibility of producing succinate aerobically using sucrose as the carbon source.
Related JoVE Video
Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases.
Metab. Eng.
PUBLISHED: 03-15-2011
Show Abstract
Hide Abstract
Microbial biosynthesis of fatty acid-like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Free fatty acids can be produced by introducing an acyl-acyl carrier protein thioesterase gene into Escherichia coli. The presence of the acyl-ACP thioesterase will break the fatty acid elongation cycle and release free fatty acid. Depending on their sequence similarity and substrate specificity, class FatA thioesterase is active on unsaturated acyl-ACPs and class FatB prefers saturated acyl group. Different acyl-ACP thioesterases have different degrees of chain length specificity. Although some of these enzymes have been characterized from a number of sources, information on their ability to produce free fatty acid in microbial cells has not been extensively examined until recently. In this study, we examined the effect of the overexpression of acyl-ACP thioesterase genes from Diploknema butyracea, Gossypium hirsutum, Ricinus communis and Jatropha curcas on free fatty acid production. In particular, we are interested in studying the effect of different acyl-ACP thioesterase on the quantities and compositions of free fatty acid produced by an E. coli strain ML103 carrying these constructs. It is shown that the accumulation of free fatty acid depends on the acyl-ACP thioesterase used. The strain carrying the acyl-ACP thioesterase gene from D. butyracea produced approximately 0.2g/L of free fatty acid while the strains carrying the acyl-ACP thioesterase genes from R. communis and J. curcas produced the most free fatty acid at a high level of more than 2.0 g/L at 48 h. These two strains accumulated three major straight chain free fatty acids, C14, C16:1 and C16 at levels about 40%, 35% and 20%, respectively.
Related JoVE Video
Effect of culture operating conditions on succinate production in a multiphase fed-batch bioreactor using an engineered Escherichia coli strain.
Appl. Microbiol. Biotechnol.
PUBLISHED: 03-13-2011
Show Abstract
Hide Abstract
A metabolically engineered Escherichia coli strain SBS550MG (pHL413) was used in this study to investigate the impact of various culture operating conditions for improving the specific succinate production rate for better final titer while maintaining the theoretical succinate yield on glucose in multiphase fed-batch cultures. Previously, we reported that changes in the level of aeration during the cell growth phase significantly modified gene expression profiles and metabolic fluxes in this system (Martinez et al. 2010). Based on these observations, the examination of culture conditions was mainly focused on the aerobic growth phase. It was found that 2-5 h of low dissolved oxygen culture during the aerobic phase improves cell productivity, but pH control during the aerobic phase was not favorable for the system. Cell viability has been identified as a major limiting factor for succinate production. Supplementing LB medium and betaine, an anti-osmotic stress reagent, did not improve cell activity. A higher succinate titer (537.8 mM) using the current metabolic engineering E. coli strain was achieved, which can potentially be improved further by increasing cell viability.
Related JoVE Video
Culture conditions impact on succinate production by a high succinate producing Escherichia coli strain.
Biotechnol. Prog.
PUBLISHED: 01-27-2011
Show Abstract
Hide Abstract
This work aimed to identify the key operational factors that significantly affect succinate production by the high succinate producing Escherichia coli strain SBS550MG (pHL413), which bears mutations inactivating genes adhE ldhA iclR ackpta::Cm(R) and overexpresses the pyruvate carboxylase from Lactococcus lactis. The considered factors included glucose concentration, cell density, CO(2) concentration in the gas stream, pH, and temperature. The results showed that high glucose concentrations inhibited succinate production and that there is a compromise between the total succinate productivity and succinate specific productivity, where the total productivity increased with the increase in cell density and the specific productivity decreased with cell density, probably due to mass transfer limitation. On the other hand, a CO(2) concentration of 100% in the gas stream showed the highest specific succinate productivity, probably by favoring pyruvate carboxylation, increasing the OAA pool that later is converted into succinate. A full factorial design of experiments was applied to analyze the pH and temperature effects on succinate production in batch bioreactors, where succinate yield was not significantly affected by either temperature (37 to 43°C) or pH (6.5 to 7.5). Additionally, the temperature effect on succinate productivity and titer was not significant, in the range tested. On the other hand, a pH of 6.5 showed very low productivity, whereas pH values of 7.0 and 7.5 resulted in significantly higher specific productivities and higher titers. The increase on pH value from 7.0 to 7.5 did not show significant improvement. Then, pH 7.0 should be chosen because it involves a lower cost in base addition.
Related JoVE Video
Effect of sodium nitroprusside on growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root cultures.
Biotechnol. Prog.
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
Nitric oxide (NO) is known as a signaling molecule involved in elicitor-induced defense responses of plants. Sodium nitroprusside (SNP), a donor of NO, stimulates catharanthine formation in Catharanthus roseus cells.1 Two important terpenoid indole alkaloids produced in small quantities within C. roseus are vinblastine and vincristine which are being used clinically as anticancer drugs. We are interested in engineering C. roseus hairy roots to increase the production of the TIAs. The present work investigates the effects of treating different concentrations of SNP to the hairy root cultures from line LBE-6-1. The alkaloid concentrations were analyzed 9, 14, 17, 20, 23, 26, and 30 days after treatment of SNP on day 0. We also studied the transient effects of SNP treatment during the exponential phase in C. roseus hairy roots. Analysis of the results showed that treatment of 0.1-mM SNP did not affect the growth of hairy roots, whereas 1-mM SNP suppressed the growth significantly, and 10-mM SNP almost completely inhibited the growth of hairy roots. 0.1-mM SNP treatment on day 0 caused a significant increase in the concentration of serpentine, catharanthine, ajmalicine, lochnericine and tabersonine production. SNP treatment on day 12 stimulated the formation of serpentine, catharanthine, ajmalicine, hörhammericine, lochnericine and tabersonine by day 21. After the initial stimulation, serpentine, horhammericine and lochnericine concentrations returned to the basal level by day 28. Treatment of 0.1-mM SNP on day 0 caused significant decrease in the mRNA levels for TDC, ASA, STR, ORCA3, ZCT1, and Crgbf1 on day 23. Treating 0.1-mM SNP on day 12 caused decreases in the expression levels of STR, ORCA3, ZCT1, and Crgbf1 on day 21 and day 28. Compared with day 28, the mRNA transcript of ZCT1 on day 21 is about twofold higher. Expression levels of G10H increased significantly.
Related JoVE Video
Succinate production from different carbon sources under anaerobic conditions by metabolic engineered Escherichia coli strains.
Metab. Eng.
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
Succinic acid has drawn much interest as a precursor of many industrially important chemicals. Using a variety of feedstocks for the bio-production of succinic acid would be economically beneficial to future industrial processes. Escherichia coli SBS550MG is able to grow on both glucose and fructose, but not on sucrose. Therefore, we derived a SBS550MG strain bearing both the pHL413 plasmid, which contains Lactococcus lactis pycA gene, and the pUR400 plasmid, which contains the scrK, Y, A, B, and R genes for sucrose uptake and catalyzation. Succinic acid production by this modified strain and the SBS550pHL413 strain was tested on fructose, sucrose, a mixture of glucose and fructose, a mixture of glucose, fructose and sucrose, and sucrose hydrolysis solution. The modified strain can produce succinic acid efficiently from all combinations of different carbon sources tested with minimal byproduct formation and with high molar succinate yields close to that of the maximum theoretic values. The molar succinic acid yield from fructose was the highest among the carbon sources tested. Using the mixture of glucose and fructose as the carbon source resulted in slightly lower yields and much higher productivity than using fructose alone. Fermenting sucrose mixed with fructose and glucose gave a 1.76-fold higher productivity than that when sucrose was used as the sole carbon source. Using sucrose pretreated with sulfuric acid as carbon source resulted in a similar succinic acid yield and productivity as that when using the mixture of sucrose, fructose, and glucose. The results of the effect of agitation rate in aerobic phase on succinate production showed that supplying large amount of oxygen in aerobic phase resulted in higher productions of formate and acetate, and therefore lower succinate yield. This study suggests that fructose, sucrose, mixture of glucose and fructose, mixture of glucose, fructose and sucrose, or sucrose hydrolysis solution could be used for the economical and efficient production of succinic acid by our metabolic engineered E. coli strain.
Related JoVE Video
Heterologous pyc gene expression under various natural and engineered promoters in Escherichia coli for improved succinate production.
J. Biotechnol.
PUBLISHED: 01-14-2011
Show Abstract
Hide Abstract
In this study, the expression level of the pyc gene from Lactococcus lactis was fine tuned to improve succinate production in Escherichia coli SBS550MG. IPTG induction in the cultures of SBS550MG with pHL413, a positive control plasmid previously constructed (Sanchez et al., 2005), gave drastically decreased PYC activity and succinate yield. We constructed several plasmids for the expression of pyc to change copy number and variant promoters. Among the constructs, as compared to pHL413, the PYC activity dropped significantly with the Plac, Ptac, Ptrc or native Ppyc promoters in medium or high copy vectors, which resulted in a decrease in succinate yield. Three constructs pThio12, pHL413-Km, and pHL413-Km(lacIq-)N showed considerable PYC activity and improved succinate production in E. coli SBS550MG. The native Ppyc promoter was also modified in order to vary pyc expression levels by site-directed mutagenesis of the -10, -35, -44 regions, and the spacer regions between -10 to -35 and -35 to -44 regions. Out of 9 native promoter variants, the MIII variant resulted in a 20% increase in PYC activity, and improved succinate yield in SBS550MG. We also determined the copy number and stability of pHL413 and pHL413-Km. The two plasmids showed roughly the same copy number, but the pHL413-Km plasmid was relatively more stable. This study provides more understanding of the plasmid characteristics and fine tuning of the expression level of pyc for optimization of the succinate production processes.
Related JoVE Video
The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots.
Metab. Eng.
PUBLISHED: 06-16-2010
Show Abstract
Hide Abstract
The terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus produces two important anticancer drugs, vinblastine and vincristine, in very low yields. This study focuses on overexpressing several key genes in the upper part of the TIA pathway in order to increase flux toward downstream metabolites within hairy root cultures. Specifically, we constructed hairy root lines with inducible overexpression of 1-deoxy-D-xylulose synthase (DXS) or geraniol-10-hydroxylase (G10H). We also constructed hairy root lines with inducible expression of DXS and anthranilate synthase ? subunit (ASA) or DXS and G10H. DXS overexpression resulted in a significant increase in ajmalicine by 67%, serpentine by 26% and lochnericine by 49% and a significant decrease in tabersonine by 66% and hörhammericine by 54%. Co-overexpression of DXS and G10H caused a significant increase in ajmalicine by 16%, lochnericine by 31% and tabersonine by 13%. Likewise, DXS and ASA overexpression displayed a significant increase in hörhammericine by 30%, lochnericine by 27% and tabersonine by 34%. These results point to the need for overexpressing multiple genes within the pathway to increase the flux toward vinblastine and vincristine.
Related JoVE Video
Metabolic impact of the level of aeration during cell growth on anaerobic succinate production by an engineered Escherichia coli strain.
Metab. Eng.
PUBLISHED: 05-09-2010
Show Abstract
Hide Abstract
The metabolic impact of two different aeration conditions during the growth phase on anaerobic succinate production by the high succinate producer Escherichia coli SBS550MG (pHL413) was investigated. Gene expression profiles, metabolites concentrations and metabolic fluxes were analyzed. Different oxygen levels are known to induce or repress transcription, synthesis of different enzymes, or both, affecting cell metabolism and thus product yield and productivity. The succinate yield was 1.55 and 1.25 mol succinate/mol glucose, and the productivity was 1.3 and 0.9 g L(-1)h(-1)) for the low aeration experiment and high aeration experiment, respectively. Changes in the level of aeration during the cells growth phase significantly modified gene expression profiles and metabolic fluxes in this system. Pyruvate was accumulated during the anaerobic phase in the high aeration experiment, which could be explained by a lower pflAB expression during the transition time and a lower flux towards acetyl-CoA during the anaerobic phase compared to the low aeration case. The higher PflAB flux and the higher expression of genes related to the glyoxylate shunt (aceA, aceB, acnA, acnB) during the transition time, anaerobic phase, or both, improved succinate yield in the low aeration case, allowing the system to attain the maximum theoretical succinate yield for E. coli SBS550MG (pHL413).
Related JoVE Video
Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid.
Biotechnol. Prog.
PUBLISHED: 09-24-2009
Show Abstract
Hide Abstract
An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD(+). Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD(+) regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD(+) as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA-pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25 degrees C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM.
Related JoVE Video
Metabolic flux analysis of Escherichia coli creB and arcA mutants reveals shared control of carbon catabolism under microaerobic growth conditions.
J. Bacteriol.
PUBLISHED: 06-26-2009
Show Abstract
Hide Abstract
Escherichia coli has several elaborate sensing mechanisms for response to availability of oxygen and other electron acceptors, as well as the carbon source in the surrounding environment. Among them, the CreBC and ArcAB two-component signal transduction systems are responsible for regulation of carbon source utilization and redox control in response to oxygen availability, respectively. We assessed the role of CreBC and ArcAB in regulating the central carbon metabolism of E. coli under microaerobic conditions by means of (13)C-labeling experiments in chemostat cultures of a wild-type strain, DeltacreB and DeltaarcA single mutants, and a DeltacreB DeltaarcA double mutant. Continuous cultures were conducted at D = 0.1 h(-1) under carbon-limited conditions with restricted oxygen supply. Although all experimental strains metabolized glucose mainly through the Embden-Meyerhof-Parnas pathway, mutant strains had significantly lower fluxes in both the oxidative and the nonoxidative pentose phosphate pathways. Significant differences were also found at the pyruvate branching point. Both pyruvate-formate lyase and the pyruvate dehydrogenase complex contributed to acetyl-coenzyme A synthesis from pyruvate, and their activity seemed to be modulated by both ArcAB and CreBC. Strains carrying the creB deletion showed a higher biomass yield on glucose compared to the wild-type strain and its DeltaarcA derivative, which also correlated with higher fluxes from building blocks to biomass. Glyoxylate shunt and lactate dehydrogenase were active mainly in the DeltaarcA strain. Finally, it was observed that the tricarboxylic acid cycle reactions operated in a rather cyclic fashion under our experimental conditions, with reduced activity in the mutant strains.
Related JoVE Video
The effects of UV-B stress on the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots.
Biotechnol. Prog.
PUBLISHED: 05-30-2009
Show Abstract
Hide Abstract
In nature, plants generate protective secondary metabolites in response to environmental stresses. Such metabolites include terpenoid indole alkaloids (TIAs), which absorb UV-B light and serve putatively to protect the plant from harmful radiation. Catharanthus roseus plants, multiple shoot cultures, and cell suspension cultures exposed to UV-B light show significant increases in the production of TIAs, including precursors to vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma. Here, the effect of UV-B light on C. roseus hairy roots was examined. Analysis of alkaloid concentrations up to 168 h after UV-B exposure shows significant increases in the concentrations of lochnericine and significant decreases in the concentration of hörhammericine over time (ANOVA, P < 0.05). Our results also indicate that increasing UV-B exposure time up to 20 min caused significant increases in lochnericine, serpentine, and ajmalicine and a decrease in hörhammericine (t-test, p < 0.05).
Related JoVE Video
Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time.
Metab. Eng.
PUBLISHED: 05-30-2009
Show Abstract
Hide Abstract
Jasmonic acid (JA) activates the transcriptional regulator ORCA3, which has a role in regulating the terpenoid indole alkaloid (TIA) pathway within Catharanthus roseus. The TIA pathway leads to the production of the anticancer drugs vinblastine and vincristine. This work explores the transient effects of overexpressing ORCA3 under the control of a glucocorticoid-inducible promoter system in C. roseus hairy roots along with the simultaneous feeding of JA. The changes in TIA metabolites and in mRNA transcripts of pathway genes and regulators were tracked for 72h. Upon induction of ORCA3 expression and elicitation with JA, ORCA3 transcripts increased 170-fold whereas ORCA3 expression caused an 89-fold increase and JA elicitation caused a 5-fold increase in ORCA3 transcripts. JA treatment caused the largest increase in TIA metabolites and transcripts of pathway genes. These transcripts displayed a transient response with the maximum expression reached between 12 and 24h. In the samples overexpressing ORCA3, the largest increase in the transcripts of ZCT1 and ZCT2 (ZCT-zinc finger-binding protein), TIA transcriptional repressors, coincided with the largest increase in ORCA3 transcripts. This counter response of transcriptional repressors may explain why the large increase in ORCA3 transcripts do not correspond with larger increases in transcripts of TIA pathway genes.
Related JoVE Video
The role of the octadecanoid pathway in the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots under normal and UV-B stress conditions.
Biotechnol. Bioeng.
PUBLISHED: 05-14-2009
Show Abstract
Hide Abstract
The octadecanoid pathway is responsible for producing jasmonic acid an important signaling molecule in plants, which controls the production of a variety of secondary metabolites. Previously the exogenous addition of jasmonic acid to Catharanthus roseus hairy roots caused an increase in terpenoid indole alkaloid (TIA) accumulation. The role of the endogenous production of jasmonic acid by the octadecanoid pathway in the production of TIAs in C. roseus hairy roots is examined. Feeding of octadecanoid pathway inhibitors suggests that the octadecanoid pathway does not actively control TIA production under normal growth conditions or during the UV-B stress response in C. roseus hairy roots.
Related JoVE Video
Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots.
Biotechnol. Bioeng.
PUBLISHED: 04-08-2009
Show Abstract
Hide Abstract
Transgenic hairy root cultures have the potential to be an industrial production platform for a variety of chemicals. This report demonstrates the long-term stability of a transgenic Catharanthus roseus hairy root line containing the inducible expression of a feedback-insensitive anthranilate synthase (AS). After 5 years in liquid culture, the presence of the inserted AS gene was confirmed by genomic PCR. The inducible expression of AS was confirmed by enzyme assay and by changes in terpenoid indole alkaloid concentrations. This report also demonstrates that it may take as long as 2 years for the metabolite profile to stabilize.
Related JoVE Video
Flavonoid glucosides from the hairy roots of Catharanthus roseus.
J. Nat. Prod.
PUBLISHED: 03-11-2009
Show Abstract
Hide Abstract
Four new flavonoid glucosides, 3,4-di-O-methylquercetin-7-O-[(4-->13)-2,6,10,14-tetramethylhexadec-13-ol-14-enyl]-beta-D-glucopyranoside (1), 4-O-methylkaempferol-3-O-[(4-->13)- 2,6,10,14-tetramethylhexadecan-13-olyl]-beta-D-glucopyranoside (2), 3,4-di-O-methylbutin-7-O-[(6-->1)-3,11-dimethyl-7-methylenedodeca-3,10-dienyl]-beta-D-glucopyranoside (3), and 4-O-methylbutin-7-O-[(6-->1)-3,11-dimethyl-7-hydroxymethylenedodecanyl]-beta-D-glucopyranoside (4), along with the three known compounds were isolated from the methanol extract of Catharanthus roseus hairy roots. Their structures were elucidated spectroscopically. The new flavonoid glucosides inhibited both MMP-9 activity and TNF-alpha production in THP-1 cells treated with lipopolysaccharide.
Related JoVE Video
Environmentally-modulated changes in fluorescence distribution in cells with oscillatory genetic network dynamics.
J. Biotechnol.
PUBLISHED: 01-06-2009
Show Abstract
Hide Abstract
We investigated the distribution of green fluorescent protein (GFP) expression levels in a population of E. coli cells expressing an artificial genetic regulatory network, known as the "repressilator". This network originally constructed by Elowitz and Leibler in 2000 consists of three cyclically-inhibiting promoter-repressor pairs. It is because of this architecture that the network has been known to oscillate at the single-cell level under certain conditions. A series of shake flask experiments were performed and analyzed using flow cytometry to test how cell populations carrying this system could be controlled extracellularly using the inducers anhydrotetracycline (aTc) and isopropyl-beta-d-thiogalactopyranoside (IPTG). With variation of [aTc], it exhibits a novel bi-threshold behavior, such that the entire culture reaches one of three steady states at a quasi-time-invariant "reference state." Also, there is significant hysteresis. Transiently, the middle state shows damping oscillations, while the low and high states show a stable steady state. The addition of IPTG serves to fine-tune the characteristics of the aTc-only expression, lowering the average and coefficient of variation (CV) of the distributions, and possibly perturbing the network to a different state. However, in modeling this system, the multiplicity and bi-threshold behavior are not theoretically possible according to the designed interactions. In order to explain this discrepancy, we hypothesize that one or more of the repressors have a significant nonspecific interaction with a promoter that does not contain its operator site. The new modeling results incorporating these extra interactions qualitatively match our experimental findings. After constructing plasmids to test these hypotheses, we discover that at least four of these interactions exist, which can create the low and high states and multiplicity seen experimentally. This genetic architecture has flexibility in its behavior that has not been demonstrated before, and the combination of experiment and modeling enlightened our understanding of the molecular interactions driving the networks behavior, leading us to discover the significance of nonspecific interactions.
Related JoVE Video
Production of succinic acid by engineered E. coli strains using soybean carbohydrates as feedstock under aerobic fermentation conditions.
Bioresour. Technol.
Show Abstract
Hide Abstract
Escherichia coli strains HL2765 and HL27659k harboring pRU600 and pKK313 were examined for succinate production under aerobic conditions using galactose, sucrose, raffinose, stachyose, and mixtures of these sugars extracted from soybean meal and soy solubles. HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 87mM and 98mM hexose of soybean meal extract and produced 83mM and 95mM succinate, respectively. While using soy solubles extract, HL2765(pKK313)(pRU600) and HL27659k(pKK313)(pRU600) consumed 160mM and 187mM hexose and produced 158mM and 183mM succinate, respectively. Succinate yield of HL2765(pKK313)(pRU600) was low as compared to that of HL27659k(pKK313)(pRU600) while using acid hydrolysate of soybean meal or soy solubles extracts. Maximum succinate production of 312mM with a molar yield of 0.82mol/mol hexose was obtained using soy solubles hydrolysate by HL27659k(pKK313)(pRU600). This study demonstrated the use of soluble carbohydrates of the renewable feedstock, soybean as an inexpensive carbon source to produce succinate by fermentation.
Related JoVE Video
Synthesis of methyl ketones by metabolically engineered Escherichia coli.
J. Ind. Microbiol. Biotechnol.
Show Abstract
Hide Abstract
Methyl ketones are a group of highly reduced platform chemicals with widespread applications in the fragrance, flavor and pharmacological industries. Current methods for the industrial production of methyl ketones include oxidation of hydrocarbons, but recent advances in the characterization of methyl ketone synthases from wild tomato have sparked interest towards the development of microbial platforms for the industrial production of methyl ketones. A functional methyl ketone biosynthetic pathway was constructed in Escherichia coli by over-expressing two genes from Solanum habrochaites: shmks2, encoding a 3-ketoacyl-ACP thioesterase, and shmks1, encoding a beta-decarboxylase. These enzymes enabled methyl ketone synthesis from 3-ketoacyl-ACP, an intermediate in the fatty acid biosynthetic cycle. The production of 2-nonanone, 2-undecanone, and 2-tridecanone by MG1655 pTH-shmks2-shmks1 was initially detected by nuclear magnetic resonance and gas chromatography-mass spectrometry analyses at levels close to 6 mg/L. The deletion of major fermentative pathways leading to ethanol (adhE), lactate (ldhA), and acetate (pta, poxB) production allowed for the carbon flux to be redirected towards methyl ketone production, doubling total methyl ketone concentration. Variations in methyl ketone production observed under different working volumes in flask experiments led to a more detailed analysis of the effects of oxygen availability on methyl ketone concentration in order to determine optimal levels of oxygen. The methyl ketone concentration achieved with MG1655 ?adhE ?ldhA ?poxB ?pta pTrcHis2A-shmks2-shmks1, the best performer strain in this study, was approximately 500 mg/L, the highest reported for an engineered microorganism. Through the establishment of optimal operating conditions and by executing rational metabolic engineering strategies, we were able to increase methyl ketone concentrations by almost 75-fold from the initial confirmatory levels.
Related JoVE Video
Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis.
Metab. Eng.
Show Abstract
Hide Abstract
Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.