Crystal structure of the ω‐aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with other class III amino‐ transferases that have biotechnological potential

Apart from their crucial role in metabolism, pyridoxal 5′‐phosphate (PLP)‐dependent aminotransferases (ATs) constitute a class of enzymes with increasing application in industrial biotechnology. To provide better insight into the structure‐function relationships of ATs with biotechnological potential we performed a fundamental bioinformatics analysis of 330 representative sequences of pro‐ and eukaryotic Class III ATs using a structure‐guided approach. The calculated phylogenetic maximum likelihood tree revealed six distinct clades of which the first segregates with a very high bootstrap value of 92%. Most enzymes in this first clade have been functionally well characterized, whereas knowledge about the natural functions and substrates of enzymes in the other branches is sparse. Notably, in those clades 2‐6 members of the peculiar class of ω‐ATs prevail, many of which have proven useful for the preparation of chiral amines or artificial amino acids. One representative is the ω‐AT from Paracoccus denitrificans (PD ω‐AT) which catalyzes, for example, the transamination in a novel biocatalytic process for the production of L ‐homoalanine from L ‐threonine. To gain structural insight into this important enzyme, its X‐ray analysis was carried out at a resolution of 2.6 Å, including the covalently bound PLP as well as 5‐aminopentanoate as a putative amino donor substrate. On the basis of this crystal structure in conjunction with our phylogenetic analysis, we have identified a generic set of active site residues of ω‐ATs that are associated with a strong preference for aromatic substrates, thus guiding the discovery of novel promising enzymes for the biotechnological production of corresponding chiral amines. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Necessary and sufficient conditions for the asymmetric synthesis of chiral amines using ω‐aminotransferases

The half reactions of ω‐aminotransferase (ω‐AT) from Vibrio fluvialis JS17 (ω‐ATVf) were carried out using purified pyridoxal 5′‐phosphate‐enzyme (PLP‐Enz) and pyridoxamine 5′‐phosphate‐enzyme (PMP‐Enz) complexes to investigate the relative activities of substrates. In the reaction generating PMP‐Enz from PLP‐Enz using L ‐alanine as an amine donor, L ‐alanine showed about 70% of the initial reaction rate of (S)‐α‐methylbenzylamine ((S)‐α‐MBA). However, in the subsequent half reaction recycling PLP‐Enz from PMP‐Enz using acetophenone as an amine acceptor, acetophenone showed nearly negligible reactivity compared to pyruvate. These results indicate that the main bottleneck in the asymmetric synthesis of (S)‐α‐MBA lies not in the amination of PLP by alanine, but in the amination of acetophenone by PMP‐Enz, where conformational restraints of the enzyme structure is likely to be the main reason for limiting the amine group transfer from PMP‐Enz to acetophenone. Based upon those half reaction experiments using the two amino acceptors of different activity, it appears that the relative activities of the two amine donors and the two acceptors involved in the ω‐AT reactions can roughly determine the asymmetric synthesis yield of the target chiral amine compound. Predicted conversion yields of several target chiral amines were calculated and compared with the experimental conversion yields. Approximately, a positive linear correlation (Pearson's correlation coefficient = 0.92) was observed between the calculated values and the experimental conversion yields. To overcome the low (S)‐α‐MBA productivity of ω‐ATVf caused by the possible disadvantageous structural constraints for acetophenone, new ω‐ATs showing higher affinity to benzene ring of acetophenone than ω‐ATVf were computationally screened using comparative modeling and protein‐ligand docking. ω‐ATs from Streptomyces avermitilis MA‐4680 (SAV2612) and Agrobacterium tumefaciens str. C58 (Atu4761) were selected, and the two screened ω‐ATs showed higher asymmetric synthesis reaction rate of (S)‐α‐MBA and lower (S)‐α‐MBA degradation reaction rate than ω‐ATVf. To verify the higher conversion yield of the variants of ω‐ATs, the reaction with 50 mM acetophenone and 50 mM alanine was performed with coupling of lactate dehydrogenase and two‐phase reaction system. SAV2612 and Atu4761 showed 70% and 59% enhanced yield in the synthesis of (S)‐α‐MBA compared to that of ω‐ATVf, respectively. Biotechnol. Bioeng. 2011;108: 253–263. © 2010 Wiley Periodicals, Inc.     

Sodium balance in the green turtle,Chelonia mydas, in seawater and freshwater

Summary Sodium balance in the green turtle,Chelonia mydas, has been investigated in both seawater and freshwater. In seawater the unidirectional Na efflux is 131 M · 100 g^–1 · h^–1, over 90% of which is via the head region and less than 5% each is via the cloaca and the integument. After 17 days in freshwater the efflux of Na has declined by 97% and the majority is via the cloaca. The integumental efflux in freshwater is less than that in seawater indicating that a change in skin permeability, trans-skin electrical potential or pattern of blood flow has taken place. Although there are indications that Na is actively extracted from freshwater by the green turtle, this species faces a net loss of Na of the medium and the blood Na concentration falls significantly. When transferred from seawater to freshwater the turtle's orbital salt gland is turned off within 25 h after transfer. The salt gland does not become functional until 400 h after transfer from freshwater to seawater.     

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml^?1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml^?1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

     

Trade‐offs in osmoregulation and parallel shifts in molecular function follow ecological transitions to freshwater in the Alewife

Adaptation to freshwater may be expected to reduce performance in seawater because these environments represent opposing selective regimes. We tested for such a trade‐off in populations of the Alewife (Alosa pseudoharengus). Alewives are ancestrally anadromous, and multiple populations have been independently restricted to freshwater (landlocked). We conducted salinity challenge experiments, whereby juvenile Alewives from one anadromous and multiple landlocked populations were exposed to freshwater and seawater on acute and acclimation timescales. In response to acute salinity challenge trials, independently derived landlocked populations varied in the degree to which seawater tolerance has been lost. In laboratory‐acclimation experiments, landlocked Alewives exhibited improved freshwater tolerance, which was correlated with reductions in seawater tolerance and hypo‐osmotic balance, suggesting that trade‐offs in osmoregulation may be associated with local adaptation to freshwater. We detected differentiation between life‐history forms in the expression of an ion‐uptake gene (NHE3), and in gill Na⁺/K⁺‐ATPase activity. Trade‐offs in osmoregulation, therefore, may be mediated by differentiation in ion‐uptake and salt‐secreting pathways.     

Expansion of the ω‐oxidation system AlkBGTL of Pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono‐esterified dicarboxylic acid production in E. coli

The AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U g_cdw^?1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol^?1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.     

Identification of novel transaminases from a 12‐aminododecanoic acid‐metabolizing Pseudomonas strain

A Pseudomonas species [Pseudomonas sp. strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12‐aminododecanoic acid, the constituent building block of homo‐nylon‐12, as a sole nitrogen source. Growth of Pseudomonas sp. strain AAC could also be supported using a range of additional ω‐amino alkanoates. This metabolic function was shown to be most probably dependent upon one or more transaminases (TAs). Fourteen genes encoding putative TAs were identified from the genome of Pseudomonas sp. AAC. Each of the 14 genes was cloned, 11 of which were successfully expressed in Escherichia coli and tested for activity against 12‐aminododecanoic acid. In addition, physiological functions were proposed for 9 of the 14 TAs. Of the 14 proteins, activity was demonstrated in 9, and of note, 3 TAs were shown to be able to catalyse the transfer of the ω‐amine from 12‐aminododecanoic acid to pyruvate. Based on this study, three enzymes have been identified that are promising biocatalysts for the production of nylon and related polymers.     

Histological and enzyme histochemical changes found in the renal tubules of eels transferred from freshwater to seawater

Renal tubules of the eel,Anguilla japonica, transferred from freshwater to seawater were examined histologically and histochemically. The epithelial cells of renal tubules in eels adapted to seawater for 10 days were less interdigitated in comparison with those in freshwater. The poor interdigitation of epithelial cells seems to be a structure relating to high water permeability in the renal tubules of eels in seawater. Newly formed nephrons and degenerated ones were especially recognized in eels adapted to seawater for 2 and 4 days. These findings indicate that some of the nephrons equipped with poorly interdigitated epithelial cells are produced newly within a few days after transfer from freshwater to seawater. No significant difference was encountered in the activities and localizations of all enzymes examined between the renal tubules of freshwater and seawater eels. The enzyme activities and localizations did not reflect the physiological changes in the renal tubules of eels adapted from freshwater to seawater.     

Sulfate inhibition of molybdenum-dependent nitrogen fixation by planktonic cyanobacteria under seawater conditions: a non-reversible effect

The trace element molybdenum is a central component of several enzymes essential to bacterial nitrogen metabolism, including nitrogen fixation. Despite reasonably high dissolved concentrations (for a trace metal) of molybdenum in seawater, evidence suggests that its biological reactivity and availability are lower in seawater than in freshwater. We have previously argued that this difference is related to an inhibition in the uptake of molybdate (the thermodynamically stable form of molybdenum in oxic natural waters) by sulfate, a stereochemically similar ion. Low molybdenum availability may slow the growth rate of nitrogen-fixing cyanobacteria, and in combination with an ecological control such as grazing by zooplankton, keep fixation rates very low in even strongly nitrogen-limited coastal marine ecosystems. Here we present results from a seawater mesocosm experiment where the molybdenum concentration was increased 10-fold under highly nitrogen-limited conditions. The observed effects on nitrogen-fixing cyanobacterial abundance and nitrogen-fixation inputs were much smaller than expected. A follow-up experiment with sulfate and molybdenum additions to freshwater microcosms showed that sulfate (at seawater concentrations) greatly reduced nitrogen fixation by cyanobacteria and that additions of molybdenum to the levels present in the seawater mesocosm experiment only slightly reversed this effect. In light of these results, we re-evaluated our previous work on the uptake of radio-labeled molybdenum by lake plankton and by cultures of heterocystic cyanobacteria. Our new interpretation indicates that sulfate at saline estuarine levels (>8–10 mM) up to seawater (28 mM) concentrations does inhibit molybdenum assimilation. However, the maximum molybdenum uptake rate (V _max) was a function of the sulfate concentration, with lower V _max values at higher sulfate levels. This indicates that this inhibition is not fully reversed at some saturating level of molybdenum, as assumed in a simple competitive inhibition model. A multi-enzyme, mixed kinetics model with two or more uptake enzyme systems activated in response to the environmental sulfate and molybdate conditions may better explain the repressive effect of sulfate on Mo-mediated processes such as nitrogen fixation.     

Immune responses of Litopenaeus vannamei to thermal stress: a comparative study of shrimp in freshwater and seawater conditions

The effects of hypothermal (22–16?°C) and hyperthermal (22–28?°C) stress on the immune system responses of the shrimp Litopenaeus vannamei cultured in either freshwater or seawater were measured and compared. The following immune system indicators were measured for comparison: total hemocyte count (THC), activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase (SOD), and malondialdehyde (MDA) content. Thermal stress significantly decreased THC in both freshwater and seawater shrimp within 6–12?h (P?0.05). After hypothermal stress, all shrimp had a significantly lower THC level than their prechallenge levels (P?0.05). Under both types of thermal stress, the activity of PO, NOS, and SOD first increased and then decreased. After 48?h of thermal stress, shrimp PO and NOS activity decreased in both freshwater and seawater. After 48?h of thermal stress, the reduction in the SOD activity in the hemolymph of freshwater shrimp was greater than that in seawater shrimp. During exposure to stress, the MDA content in freshwater shrimp was significantly higher than in seawater shrimp, which demonstrated that lipids in freshwater shrimp were more susceptible to peroxidation than those in seawater shrimp, particularly at low temperatures. Large temperature fluctuations, particularly sudden cooling, should be avoided when rearing L. vannamei because of high rates of lipid peroxidation and decreased immunity. These effects are more marked in freshwater than in seawater.     

ω‐Turn: A novel β‐turn mimic in globular proteins stabilized by main‐chain to side‐chain CH···O interaction

Mimicry of structural motifs is a common feature in proteins. The 10‐membered hydrogen‐bonded ring involving the main‐chain C?O in a β‐turn can be formed using a side‐chain carbonyl group leading to Asx‐turn. We show that the N? H component of hydrogen bond can be replaced by a C^γ‐H group in the side chain, culminating in a nonconventional C? H···O interaction. Because of its shape this β‐turn mimic is designated as ω‐turn, which is found to occur ～three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C? H···O interaction occurring between the terminal residues, constraining the torsion angles ?_{i + 1}, ψ_{i + 1}, ?_{i + 2} and χ′_{1(i + 2)} (using the interacting C^γ atom). Based on these angles there are two types of ω‐turns, each of which can be further divided into two groups. C^β‐branched side‐chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal‐binding sites. N‐linked glycosylation occurs at the consensus pattern Asn‐Xaa‐Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω‐turn, which may be the recognition site for protein modification. Location between two β‐strands is the most common occurrence in protein tertiary structure, and being generally exposed ω‐turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2015; 83:203–214. © 2014 Wiley Periodicals, Inc.     

Whole‐cell biocatalytic of Bacillus cereus WZZ006 strain to synthesis of indoxacarb intermediate: (S)‐5‐Chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester

In this study, a newly isolated strain screened from the indoxacarb‐rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester was obtained by bio‐enzymatic resolution. After the 36‐hour hydrolysis in 50‐mM racemic substrate under the optimized reaction conditions, the e.e._s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high‐level ability to produce (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.     

盐分对河口淡水、微咸水沼泽湿地土壤甲烷产生潜力的影响

海平面上升导致河口区盐水入侵现象日益明显,深刻影响着河口潮汐淡水、微咸水湿地生物地球化学循环。采集闽江河口区淡水、微咸水短叶茳芏潮汐沼泽湿地表层土样,室内添加盐度为5,10,15,21 g/L的人造海水、Na Cl溶液及盐度为0的去离子水,通过室内泥浆厌氧培养试验,对比研究海水和Na Cl溶液对淡水、微咸水沼泽湿地土壤甲烷产生潜力的影响。与对照相比,1—12 d培养期内4个盐度的海水处理均显著抑制河口淡水、微咸水沼泽湿地甲烷产生潜力,抑制率在93%以上,盐度10—21 g/L的3个海水处理对于河口淡水、微咸水沼泽湿地甲烷产生潜力的抑制效应无显著差异。Na Cl溶液只有在盐度达到15和21 g/L才显著抑制淡水、微咸水沼泽湿地甲烷产生潜力,且抑制率最多为80.9%,盐度为5、10 g/L的Na Cl溶液对淡水、微咸水沼泽湿地甲烷产生潜力的抑制作用不显著,抑制率多小于30%。伴随着盐水入侵而发生的硫酸盐还原作用及离子胁迫作用对河口淡水、微咸水沼泽湿地甲烷产生具有显著的抑制效应。     

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

Abstract

This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.     

The ω‐transaminase engineering database (oTAED): A navigation tool in protein sequence and structure space

The ω‐Transaminase Engineering Database (oTAED) was established as a publicly accessible resource on sequences and structures of the biotechnologically relevant ω‐transaminases (ω‐TAs) from Fold types I and IV. The oTAED integrates sequence and structure data, provides a classification based on fold type and sequence similarity, and applies a standard numbering scheme to identify equivalent positions in homologous proteins. The oTAED includes 67 210 proteins (114 655 sequences) which are divided into 169 homologous families based on global sequence similarity. The 44 and 39 highly conserved positions which were identified in Fold type I and IV, respectively, include the known catalytic residues and a large fraction of glycines and prolines in loop regions, which might have a role in protein folding and stability. However, for most of the conserved positions the function is still unknown. Literature information on positions that mediate substrate specificity and stereoselectivity was systematically examined. The standard numbering schemes revealed that many positions which have been described in different enzymes are structurally equivalent. For some positions, multiple functional roles have been suggested based on experimental data in different enzymes. The proposed standard numbering schemes for Fold type I and IV ω‐TAs assist with analysis of literature data, facilitate annotation of ω‐TAs, support prediction of promising mutation sites, and enable navigation in ω‐TA sequence space. Thus, it is a useful tool for enzyme engineering and the selection of novel ω‐TA candidates with desired biochemical properties.     

Green synthesis of enantiopure (S)‐1‐(benzofuran‐2‐yl)ethanol by whole‐cell biocatalyst

Optically active aromatic alcohols are valuable chiral building blocks of many natural products and chiral drugs. Lactobacillus paracasei BD87E6, which was isolated from a cereal‐based fermented beverage, was shown as a biocatalyst for the bioreduction of 1‐(benzofuran‐2‐yl) ethanone to (S)‐1‐(benzofuran‐2‐yl) ethanol with highly stereoselectivity. The bioreduction conditions were optimized using L. paracasei BD87E6 to obtain high enantiomeric excess (ee) and conversion. After optimization of the bioreduction conditions, it was shown that the bioreduction of 1‐(benzofuran‐2‐yl)ethanone was performed in mild reaction conditions. The asymmetric bioreduction of the 1‐(benzofuran‐2‐yl)ethanone had reached 92% yield with ee of higher than 99.9% at 6.73 g of substrate. Our study gave the first example for enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol by a biological green method. This process is also scalable and has potential in application. In this study, a basic and novel whole‐cell mediated biocatalytic method was performed for the enantiopure production of (S)‐1‐(benzofuran‐2‐yl)ethanol in the aqueous medium, which empowered the synthesis of a precious chiral intermediary process to be converted into a sophisticated molecule for drug production.     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.