Yield Of ALA Research Articles

Metabolic engineering of Escherichia coli for the production of 5-aminolevulinic acid based on combined metabolic pathway modification and reporter-guided mutant selection (RGMS)

Background5-Aminolevulinic acid (ALA) recently received much attention due to its potential application in many fields such as medicine, nutrition and agriculture. Metabolic engineering is an efficient strategy to improve microbial production of 5-ALA.ResultsIn this study, an ALA production strain of Escherichia coli was constructed by rational metabolic engineering and stepwise improvement. A metabolic strategy to produce ALA directly from glucose in this recombinant E. coli via both C4 and C5 pathways was applied herein. The expression of a modified hemARS gene and rational metabolic engineering by gene knockouts significantly improved ALA production from 765.9 to 2056.1 mg/L. Next, we tried to improve ALA production by RGMS-directed evolution of eamA gene. After RGMS, the ALA yield of strain A2-ASK reached 2471.3 mg/L in flask. Then, we aimed to improve the oxidation resistance of cells by overexpressing sodB and katE genes and ALA yield reached 2703.8 mg/L. A final attempt is to replace original promoter of hemB gene in genome with a weaker one to decrease its expression. After 24 h cultivation, a high ALA yield of 19.02 g/L was achieved by 108-ASK in a 5 L fermenter.ConclusionsThese results suggested that an industrially competitive strain can be efficiently developed by metabolic engineering based on combined rational modification and optimization of gene expression.

The tree peony DREB transcription factor PrDREB2D regulates seed α-linolenic acid accumulation.

α-Linolenic acid (ALA), an essential fatty acid (FA) for human health, serves as the precursor of 2 nutritional benefits, docosahexaenoic acid and eicosapentaenoic acid, and can only be obtained from plant foods. We previously found that phospholipid:diacylglycerol acyltransferase 2 (PrPDAT2) derived from ALA-rich tree peony (Paeonia rockii) can promote seed ALA accumulation. However, the regulatory mechanism underlying its promoting effect on ALA accumulation remains unknown. Here, we revealed a tree peony dehydration-responsive element binding transcription factor, PrDREB2D, as an upstream regulator of PrPDAT2, which is involved in regulating seed ALA accumulation. Our findings demonstrated that PrDREB2D serves as a nucleus-localized transcriptional activator that directly activates PrPDAT2 expression. PrDREB2D altered the FA composition in transient overexpression Nicotiana benthamiana leaves and stable transgenic Arabidopsis (Arabidopsis thaliana) seeds. Repressing PrDREB2D expression in P. rockii resulted in decreased PrPDAT2 expression and ALA accumulation. In addition, PrDREB2D strengthened its regulation of ALA accumulation by recruiting the cofactor ABA-response element binding factor PrABF2b. Collectively, the study findings provide insights into the mechanism of seed ALA accumulation and avenues for enhancing ALA yield via biotechnological manipulation.

Integrative lipidomics profile uncovers the mechanisms underlying high-level α-linolenic acid accumulation in Paeonia rockii seeds.

Tree peony (Paeonia rockii) is an excellent woody oilseed crop, known for its high α-linolenic acid (ALA, ~45%) content, which is of great value for human health. However, the mechanisms underlying this high-level ALA accumulation in tree peony seeds are poorly understood. In this study, we evaluated the dynamic changes in the lipidomic profile of P. rockii seeds during development. A total of 760 lipid molecules were identified in P. rockii seeds; triacylglycerol (TAG) lipid molecules showed the highest abundance and diversity, both increasing during seed development. Particularly, ALA was the predominant fatty acid at the TAG sn-3 position. We further characterized two diacylglycerol acyltransferase (DGAT) genes and three phospholipid:diacylglycerol acyltransferase (PDAT) genes involved in the transfer of fatty acids to the TAG sn-3 position. Gene expression and subcellular localization analyses suggested that PrDGATs and PrPDATs may function as endoplasmic reticulum-localized proteins in seed TAG biosynthesis. In vitro functional complementation analysis showed different substrate specificities, with PrPDAT2 having a specific preference for ALA. Multiple biological assays demonstrated that PrDGAT1, PrDGAT2, PrPDAT1-2, and PrPDAT2 promote oil synthesis. Specifically, PrPDAT2 leads to preferential ALA in the oil. Our findings provide novel functional evidence of the roles of PrDGAT1 and PrPDAT2, which are potential targets for increasing the ALA yield in tree peony and other oilseed crops.

Urea complexation combined with rapid preparative reversed-phase liquid chromatography to separate α-linolenic acid from perilla seed oil: Purity, yield, and oxidation stability

Perilla seed oil (PSO) is a major source of α-linolenic acid (ALA). In order to better separate ALA from PSO and to improve its purity and yield, a purification method for ALA that combines urea complexation (UC) with rapid preparative reversed-phase liquid chromatography (RP-RPLC) has been studied. Within the selected ranges of conditions, four one-factor experiments were found to have significant effects on the purity and yield of ALA from PSO. A response surface methodology (RSM) has been applied to optimize UC for separating and purifying ALA from PSO, which yielded optimal parameters of 95% ethanol/urea (w/w) = 5:1, urea/esterified fatty acids (E-FAs) (w/w) = 2:1, complexation time 16.3 h, and complexation temperature 17 °C. The optimal purity and optimal yield of the one-time purification ALA (OTP-ALA) obtained by UC thereby reached 86.977 ± 0.082% and 34.696 ± 0.948 %, respectively. Two-time purification conditions of OTP-ALA by RP-RPLC were also determined, and the purity of the two-time purification ALA (TTP-ALA) was increased to 99.200 ± 0.061%, corresponding to a yield of 53.888 ± 0.904 %. The effect of different added amounts of α-tocopherol on the oxidation stability of TTP-ALA has also been studied. Accelerated oxidation tests showed that the addition of α-tocopherol effectively inhibited the oxidation of TTP-ALA by reducing the peroxide value (PV). of ALA separation and purification in PSO (triglycerides refers to the collective term for all fatty acid glycerides in PSO). • An improved method for purification of ALA from perilla seed oil was developed. • RSM has been used to optimize the separation of ALA from perilla seed oil. • The combined use of the two methods results in ALA purity exceeding 99.2 %. • Add an appropriate amount of α-tocopherol improved the oxidation stability of ALA.

Effect of Flaxseed Supplementation on Milk and Plasma Fatty Acid Composition and Plasma Parameters of Holstein Dairy Cows.

Simple SummaryFew articles have reported the influence of whole and ground flaxseed intake on milk longer chain n-3 polyunsaturated fatty acid (PUFA) and the efficiency of diet α-linolenic acid (ALA) to convert to milk (milk ALA/ALA intake ratio). The objective of this experiment was to explore the effect of whole and ground flaxseed supplementation on the composition of n-3 polyunsaturated fatty acid (PUFA) in milk from dairy cows. This result indicated that whole and ground flaxseed supplementation can increase total n-3 PUFA. Compared with whole flaxseed, ground flaxseed supplementation showed higher content and yield of ALA in milk and milk ALA/ALA intake.The objective of this study was to determine the effect of whole flaxseed and ground flaxseed supplementation on the composition of fatty acids in plasma and milk, particularly the content of omega-3 polyunsaturated fatty acids (n-3 PUFAs). Thirty Holstein dairy cows were randomly assigned to three treatment groups. Cows were fed a total mixed ration without flaxseed (CK), 1500 g of whole flaxseed (WF), and 1500 g of ground flaxseed (GF) supplementation. There were no differences observed in dry matter intake, milk yield, energy-corrected milk, and 4% fat-corrected milk (p > 0.05). Compared with the CK group, the contents of α-linolenic acid (ALA), eicosatrienoic acid, and eicosapentaenoic acid increased in the plasma and milk WF and GF groups, and the content of docosahexaenoic acid and total n-3 PUFA was higher in GF than the other groups (p < 0.001). The ALA yield increased to 232% and 360% in WF and GF, respectively, compared to the CK group. Compared with the WF group, GF supplementation resulted in an increased milk ALA/ALA intake ratio (p < 0.001). Flaxseed supplementation increased the activity of GSH-Px and decreased the concentration of MDA in milk (p < 0.001). Plasma parameters did not differ among the treatments (p > 0.05). This result indicated that compared with the WF group, GF supplementation in the diet showed higher efficiency in increasing the total n-3 PUFA levels and the milk ALA/ALA intake ratio, and decreased the ratio of n-6 PUFAs to n-3 PUFAs in milk.

Engineering pyridoxal kinase PdxY-integrated Escherichia coli strain and optimization for high-level 5-aminolevulinic acid production

Recently, 5-aminolevulinic acid (ALA) has gained significant attention in research due to its extensive applications in agriculture, aquaculture, cosmetics, and photodynamic therapy for cancer. Microbial production of ALA is a green, high-efficient, and sustainable process. Herein, we have engineered a new strain of Escherichia coli called PdxY Integrative E. coliEnhancer (PIECE). In this strain, the super salvage pathway was fine-tuned to enhance the regeneration of pyridoxal 5′-phosphate (PLP), on which the activity of ALA synthase (ALAS) is highly dependent. The PIECE strain, harboring ALA synthase (ALAS) from Rhodobacter capsulatus, produced 1.99 g/L ALA and 5.15 g/L biomass, that was a 4.33-fold increase in ALA production and a 1.84-fold increase in biomass compared to the parent strain. The metabolic flux analysis indicated that PIECE would redirect more carbon into the tricarboxylic acid (TCA) cycle and generate more glycine and glutamate, thereby increasing the ALA and biomass. Finally, the maximum ALA yield of 8.21 g/L and a productivity of 0.228 g/L/h were obtained by using fed-batch fermentation. The results showed that PIECE was not only feasible for high-level ALA production but also had significant potential to reprogram the carbon flux in the TCA cycle to produce value-added chemicals in the future.

Nanoliposomes and Nanoemulsions Based on Chia Seed Lipids: Preparation and Characterization.

Polyunsaturated fatty acids (PUFA) are important in reducing the risk for cardiovascular, metabolic and neurodegenerative diseases. Chia (Salvia hispanica L.) seeds contain high levels of omega-3 PUFA, α-linolenic acid (ALA) in particular, and are a potential source for development of omega-3 PUFA-based products. Our objective was to obtain and characterize chia seed lipids, focusing on phospholipid fraction, and to investigate their use in the formulation of nanoemulsions (NE) and nanoliposomes (NL). Solvent-based lipid extraction was performed on the ORURO variety of chia seeds, followed by lipid composition analysis using GC and LC-MS and physico-chemical characterization of chia NL and NE. Folch extraction led to a slightly higher yield of ALA as compared to Soxhlet extraction. Lipid, phospholipid, and fatty acid composition analysis of the oil and residue revealed that the residue was rich in phospholipids; these were used to prepare NE and NL. Physico-chemical characterization showed that NE and NL were generally spherical (transmission electron microscopy), with a size of <120 nm under hydrated conditions that remained stable over 5 days. In conclusion, chia oil and phospholipid-rich residue can be used to obtain stable NL or NE using a simple method that involves spontaneous emulsification during lipid hydration, which potentially may be useful in cosmetics, pharmaceutical, and other health applications.

Optimization of Biomass and 5-Aminolevulinic Acid Production by Rhodobacter sphaeroides ATCC17023 via Response Surface Methodology.

Microbial 5-aminolevulinic acid (ALA) produced from wastewater is considered as potential renewable energy. However, many hurdles are needed to be overcome such as the regulation of key influencing factors on ALA yield. Biomass and ALA production by Rhodobacter sphaeroides was optimized using response surface methodology. The culturing medium was artificial volatile fatty acids wastewater. Three additives were optimized, namely succinate and glycine that are precursors of ALA biosynthesis, and D-glucose that is an inhibitor of ALA dehydratase. The optimal conditions were achieved by analyzing the response surface plots. Statistical analysis showed that succinate at 8.56mmol/L, glycine at 5.06mmol/L, and D-glucose at 7.82mmol/L were the best conditions. Under these optimal conditions, the highest biomass production and ALA yield of 3.55g/L and 5.49mg/g-biomass were achieved. Subsequent verification experiments at optimal values had the maximum biomass production of 3.41 ± 0.002g/L and ALA yield of 5.78 ± 0.08mg/g-biomass.

Performance, 5-aminolevulinic acid (ALA) yield and microbial population dynamics in a photobioreactor system treating soybean wastewater: Effect of hydraulic retention time (HRT) and organic loading rate (OLR)

Effects of hydraulic retention time (HRT) and influent organic loading rate (OLR) were investigated in a photobioreactor containing PNSB (Rhodobacter sphaeroides)-chemoheterotrophic bacteria to treat soybean wastewater. Pollutants removal, biomass production and ALA yield in different phases were investigated in together with functional microbial population dynamics. The results showed that proper HRT and OLR increased the photobioreactor performance including pollutants removal, biomass and ALA productions. 89.5% COD, 90.6% TN and 91.2% TP removals were achieved as well as the highest biomass production of 2655mg/L and ALA yield of 7.40mg/g-biomass under the optimal HRT of 60h and OLR of 2.48g/L/d. In addition, HRT and OLR have important impacts on PNSB and total bacteria dynamics.

Constitutive expression of RyhB regulates the heme biosynthesis pathway and increases the 5-aminolevulinic acid accumulation inEscherichia coli

In the current study, the small RNA ryhB, which regulates the metabolism of iron in Escherichia coli, was constitutively expressed in engineered E. coli DALA. The resulting strain E. coli DALRA produced 16% more 5-aminolevulinic acid (ALA) than the parent strain E. coli DALA in batch fermentation. Meanwhile, we found that addition of iron in the medium increased heme formation and reduced ALA yield, whereas the presence of iron chelator in the medium decreased heme concentration and increased the ALA production efficiency (ALA yield per OD600). The qRT-PCR analysis showed that the mRNA levels of hemB and hemH were also decreased as well as the known RyhB target genes of acnAB, sdhAB, fumA, and cydAB in E. coli DALRA. These results indicated that small RNA can be used as a tool for regulating ALA accumulation in E. coli.

Improved 5-Aminolevulinic Acid Production with Recombinant Escherichia coli by a Short-term Dissolved Oxygen Shock in Fed-batch Fermentation

5-Aminolevulinic acid (ALA) is a common precursor for tetrapyrrole compounds in all kinds of organisms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen (DO) shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombinant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase (ALAS) activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 min in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 min. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g·L−1 (72 mmol·L−1), which is the highest yield in the fermentation broth reported up to now.

D-glucose Enhanced 5-Aminolevulinic Acid Production in Recombinant Escherichia coli Culture

In this study, we introduced a new strategy, feeding D-glucose, to overproduce extracellular 5-aminolevulinic acid (ALA) in the recombinant Escherichia coli. We investigated that the D-glucose concentration is dependent on extracellular ALA production. The results indicated that increasing D-glucose concentration in bacteria culture enhanced final cell density and ALA yield and simultaneously decreased the activities of ALA synthase (ALAS) and ALA dehydratase (ALAD); then, the inhibitory effect of D-glucose on ALAS activity was relieved with the metabolism of D-glucose. when 4.0 g/L D-glucose was added at late exponential phase; 1.46 g/L ALA was achieved in shaking culture, which is 47% or 109% higher than the ALA yields with 30 mM levulinic acid of ALAD inhibitor or no inhibitor. In jar fermenter, final extracellular ALA concentration reached 3.1 g/L by feeding with D-glucose.

Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production

The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and d-glucose, d-fructose, d-xylose, d-mannose, l-arabinose, d-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM d-glucose or d-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining d-xylose as a new inhibitor for ALAD with d-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a- hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter.

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)- hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1 g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6 h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6 g/l via fed-batch fermentation.

5-Aminolevulinate production with recombinant Escherichia coli using a rare codon optimizer host strain

The 5-aminolevulinate (ALA) synthase gene (hemA) containing several codons rarely used by Escherichia coli was cloned from the genome of Rhodobacter sphaeroides and optimized in two strains of Escherichia coli: BL21(DE3) and Rosetta(DE3), which is a rare codon optimizer strain. The effects of initial isopropyl-beta-D: -thiogalactopyranoside (IPTG) concentration, induction time, and temperature on enzyme activity were studied and compared for two strains. The results indicated that the ALA synthase expressed by Rosetta(DE3)/pET-28a(+)-hemA was higher than that by BL21(DE3)/pET-28a(+)-hemA. The initial precursors, glycine and succinate, and initial glucose, which is an inhibitor for both ALA synthase and dehydratase, were observed to be the key factors affecting ALA production. ALA synthase activity was generally higher with Rosetta(DE3) than with BL21(DE3), so was ALA biosynthesis. Based on the optimal culture system using Rosetta(DE3), the yield of ALA achieved 3.8 g/l (29 mM) under the appropriate conditions in fermenter.