Components of the Phenylpropanoid Pathway in the Implementation of the Protective Effect of Sodium Nitroprusside on Wheat under Salinity.

Nitric oxide (NO) is a multifunctional, gaseous signaling molecule implicated in both physiological and protective responses to biotic and abiotic stresses, including salinity. In this work, we studied the effects of 200 µM exogenous sodium nitroprusside (SNP, a donor of NO) on the components of the phenylpropanoid pathway, such as lignin and salicylic acid (SA), and its relationship with wheat seedling growth under normal and salinity (2% NaCl) conditions. It was established that exogenous SNP contributed to the accumulation of endogenous SA and increased the level of transcription of the pathogenesis-related protein 1 (PR1) gene. It was found that endogenous SA played an important role in the growth-stimulating effect of SNP, as evidenced by the growth parameters. In addition, under the influence of SNP, the activation of phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), and peroxidase (POD), an increase in the level of transcription of the TaPAL and TaPRX genes, and the acceleration of lignin accumulation in the cell walls of roots were revealed. Such an increase in the barrier properties of the cell walls during the period of preadaptation played an important role in protection against salinity stress. Salinity led to significant SA accumulation and lignin deposition in the roots, strong activation of TAL, PAL, and POD, and suppression of seedling growth. Pretreatment with SNP under salinity conditions resulted in additional lignification of the root cell walls, decreased stress-induced endogenous SA generation, and lower PAL, TAL, and POD activities in comparison to untreated stressed plants. Thus, the obtained data suggested that during pretreatment with SNP, phenylpropanoid metabolism was activated (i.e., lignin and SA), which contributed to reducing the negative effects of salinity stress, as evidenced by the improved plant growth parameters.

Engineering a Pseudomonas taiwanensis 4-coumarate platform for production of para-hydroxy aromatics with high yield and specificity.

Aromatics are valuable bulk or fine chemicals with a myriad of important applications. Currently, their vast majority is produced from petroleum associated with many negative aspects. The bio-based synthesis of aromatics contributes to the much-required shift towards a sustainable economy. To this end, microbial whole-cell catalysis is a promising strategy allowing the valorization of abundant feedstocks derived from biomass to yield de novo-synthesized aromatics. Here, we engineered tyrosine-overproducing derivatives of the streamlined chassis strain Pseudomonas taiwanensis GRC3 for efficient and specific production of 4-coumarate and derived aromatics. This required pathway optimization to avoid the accumulation of tyrosine or trans-cinnamate as byproducts. Although application of tyrosine-specific ammonia-lyases prevented the formation of trans-cinnamate, they did not completely convert tyrosine to 4-coumarate, thereby displaying a significant bottleneck. The use of a fast but unspecific phenylalanine/tyrosine ammonia-lyase from Rhodosporidium toruloides (RtPAL) alleviated this bottleneck, but caused phenylalanine conversion to trans-cinnamate. This byproduct formation was greatly reduced through the reverse engineering of a point mutation in prephenate dehydratase domain-encoding pheA. This upstream pathway engineering enabled efficient 4-coumarate production with a specificity of >95% despite using an unspecific ammonia-lyase, without creating an auxotrophy. In shake flask batch cultivations, 4-coumarate yields of up to 21.5% (Cmol/Cmol) from glucose and 32.4% (Cmol/Cmol) from glycerol were achieved. Additionally, the product spectrum was diversified by extending the 4-coumarate biosynthetic pathway to enable the production of 4-vinylphenol, 4-hydroxyphenylacetate, and 4-hydroxybenzoate with yields of 32.0, 23.0, and 34.8% (Cmol/Cmol) from glycerol, respectively.

Investigation of pectolytic and PR genes expression, quality and phytochemical contents in organic and non-organic table grapes at harvest and during storage.

The demand for organic table grapes is increasing worldwide. However, comprehensive information of quality parameters and phytochemical compounds in organically grown fruit remain unclear. Furthermore, table grapes are perishable and postharvest quality retention and waste prevention is very important. In this study we have compared the differences between organic and non-organic table grapes in terms of phytochemical compounds and quality parameters as well as the changes in the expression levels of pathogen related and lytic genes during storage. Organic fruit showed higher levels of phenolics, flavonoids, caffeic acid, hydrogen peroxide, protein content, antioxidant and anti-stress enzymes and total antioxidant activities at harvest and during storage. Although, the expression levels of polygalactronases, pectin methyl esterase, chitinase and glucanase genes was lower in organically grown table grapes at harvest, but the expression of all these genes was significantly increased during cold storage. After 60 days of cold storage the expression levels of pectin methyl esterase, chitinase and glucanase genes was significantly higher than the conventionally grown grape berries in organic ones. The highest expression of polygalacturonase was recorded in organic samples after 30 days of storage. There was no significant difference between the two types of table grapes for decay extension and tissue deterioration rate. The results of this study indicate that due to higher levels of phytochemicals and antioxidant compounds the organic table grapes have a higher nutritional quality. Furthermore, the increase in PR and pectolytic genes expression levels is enough for decreasing the fruit susceptibility to decay pathogens and enhancing the postharvest life of organic grapes.

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae.

BACKGROUND: Resveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts. RESULTS: To achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge. CONCLUSIONS: This study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid.

Ideal immobilization with enhanced biocatalyst activity and thermostability enables natural enzymes to serve as a powerful tool to yield synthetically useful chemicals in industry. Such an enzymatic method strategy becomes easier and more convenient with the use of genetic and protein engineering. Here, we developed a covalent programmable polyproteam of tyrosine ammonia lyases (TAL-CLEs) by fusing SpyTag and SpyCatcher peptides into the N-terminal and C-terminal of the TAL, respectively. The resulting circular enzymes were clear after the spontaneous isopeptide bonds formed between the SpyTag and SpyCatcher. Furthermore, the catalytic performance of the TAL-CLEs was measured via a synthesis sample of p-Coumaric acid. Our TAL-CLEs showed excellent catalytic efficiency, with 98.31 ± 1.14% yield of the target product-which is 4.15 ± 0.08 times higher than that of traditional glutaraldehyde-mediated enzyme aggregates. They also showed over four times as much enzyme-activity as wild-type TAL does and demonstrated good reusability, and so may become a good candidate for industrial enzymes.

Virtual screening and docking analysis of novel ligands for selective enhancement of tea (Camellia sinensis) flavonoids.

Flavour of tea is mainly contributed by a group of polyphenols - flavonoids. However, the content of flavonoid fluctuates seasonally and is found to be higher in the first flush of tea, when compared to the second flush. This disparity in the flavonoid content, and hence taste, incurs heavy economic losses to the tea plantation industry each harvest season. For our present study, four key product-specific enzymes (PAL, FNS, FLS and ANS) of the tea-specific flavonoid pathway were selected to perform molecular docking studies with specific virtually screened allosteric modulators. Results of docking analyses showed Naringenin, 2-Morpholin-4-ium-4-ylethanesulfonate, 6-C-Glucosylquercetin, 2-Oxoglutaric acid, 3,5,7,3',4'-pentahydroxyflavone to be capable of improving the spontaneity of the enzyme-substrate reactions in terms of docking score, RMSD values, and non-covalent interactions (H-bond,hydrophobic interaction, Π-stacking, salt bridge, etc.). Further, the evolutionary relationship of tea flavonoid pathway enzymes was constructed and compared with related taxa. The codon usage-based of tea flavonoid biosynthetic genes indicated the non-biasness of their nucleotide composition. Overall this study will provide a direction towards putative ligand-dependent enhancement of flavonoid content, irrespective of seasonal variation.

Discovery of Novel Tyrosine Ammonia Lyases for the Enzymatic Synthesis of p-Coumaric Acid.

p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TALclu ) has the highest affinity (Km =0.019 mm) and conversion efficiency (kcat /Km= 1631 s-1 ⋅ mm-1 ) towards l-tyrosine. The reaction conditions for two purified enzymes and their E. coli recombinant cells were optimized and p-CA yields of 2.03 g/L after 8 hours by TALclu and 2.35 g/L after 24 h by TAL from Rivularia sp. PCC 7116 (TALrpc ) in whole cells were achieved. These TALs are thus candidates for the construction of whole-cell systems to produce the flavonoid precursor p-CA.

[Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid].

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.

Effect of Heavy Metal Stress on Phenolic Compounds Accumulation in Winter Wheat Plants.

Heavy metal stress can lead to many adverse effects that inhibit cellular processes at various levels of metabolism, causing a decrease in plant productivity. In response to environmental stressors, phenolic compounds fulfill significant molecular and biochemical functions in plants. Increasing the biosynthesis of phenolic compounds in plants subjected to heavy metal stress helps protect plants from oxidative stress. A pot experiment was carried out to determine the effect of the accumulation of copper (Cu) and lead (Pb) salts at concentrations of 200, 500, and 1000 ppm on seed germination, the activity of enzymes in the phenylalanine ammonia-lyase pathway (PAL) and tyrosine ammonia-lyase (TAL), along with the total phenol and flavonoid contents in seedlings of hybrid Triticum aestivum L. (winter wheat) cultivars. The accumulation of heavy metals, especially Cu, had a negative impact on the seed germination process. The cultivar "Hyacinth" reacted most strongly to heavy metal stress, which was confirmed by obtaining the lowest values of the germination parameters. Heavy metal stress caused an increase in the activity of PAL and TAL enzymes and an increase in the accumulation of phenolic compounds. Under the influence of Cu, the highest activity was shown in cv. "Hyvento" (especially at 200 ppm) and, due to the accumulation of Pb, in cv. "Hyacinth" (1000 ppm) and cv. "Hyking" (200 ppm). The cultivar "Hyking" had the highest content of phenolic compounds, which did not increase with the application of higher concentrations of metals. In other cultivars, the highest content of total phenols and flavonoids was usually observed at the lowest concentration (200 ppm) of the tested heavy metals, Cu and Pb.

Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid / 生物工程学报

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.

Exogenous Application of Zinc to Mitigate the Salt Stress in Vigna radiata (L.) Wilczek-Evaluation of Physiological and Biochemical Processes.

Salt stress adversely affects the growth and productivity of crops. However, reports suggest that the application of various micronutrients could help the plant to cope with this stress. Hence, the objective of the study was to examine the effect of exogenous application of Zinc (Zn) on salt tolerance in Vigna radiata (L.) Wilczek (mungbean). Mungbean is considered to be an economically important crop and possess a strategic position in Southeast Asian countries for sustainable crop production. It is rich in quality proteins, minerals and vitamins. Three weeks old grown seedlings were subjected to NaCl (150 mM and 200 mM) alone or with Zn (250 µM). After 21 days of treatment, plants were harvested for investigating morphological, physiological and biochemical changes. We found that the Zn application mitigates the negative effect upon plant growth to a variable extent. This may be attributed to the increased shoot and root length, improved chlorophyll and carotenoid contents, enhanced total soluble sugar (TSS), total soluble protein (TSP) and proline accumulation, decreased H2O2 content and increased enzymatic antioxidant activities. Zn's application improved the performance of the enzymes such as phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) of the secondary metabolism, which resulted in the improvement of total phenol and flavonoids. The antioxidant activities such as 1,1diphenyl 2-picryl hydrazine (DPPH) and ferrous reducing antioxidant power assay (FRAP) of the plants also showed improved results in their salt only treatments. Furthermore, hydrogen peroxide (H2O2) and superoxide radical (SOD) scavenging activity were also improved upon the application of 250 µM zinc. Thus, Zn application in low doses offers promising potential for recovering plants suffering from salinity stress. In conclusion, we assume that zinc application improved salt tolerance in mungbean through the improvement of various physiological and photochemical processes which could prove to be useful in nutrient mediated management for crop improvement.

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT-penetrating peptide.

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg-1 •s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 µgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

[Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli].

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Characterization of two new aromatic amino acid lyases from actinomycetes for highly efficient production of p-coumaric acid.

p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from deamination of L-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 µM-1 min-1, while Sts-TAL had a much higher efficiency with a kcat/Km value of 78.3 µM-1 min-1. Both Sts-TAL and Sas-TAL can also take L-phenylalanine as the substrate to yield trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)-1, which represents the highest efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.

Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities.

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and K m value of 609 µM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)-specific activity of 80 ± 2 U/mg and K m value of 13.3 µM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

Directed evolution of tyrosine ammonia-lyase to improve the production of p-coumaric acid in Escherichia coli / 生物工程学报

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.

Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL).

Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows KM-values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules.

Rhodotorula glutinis Phenylalanine/Tyrosine Ammonia Lyase Enzyme Catalyzed Synthesis of the Methyl Ester of para-Hydroxycinnamic Acid and its Potential Antibacterial Activity.

Biotransformation of L-tyrosine methyl ester (L-TM) to the methyl ester of para- hydroxycinnamic acid (p-HCAM) using Rhodotorula glutinis yeast phenylalanine/tyrosine ammonia lyase (PTAL; EC 4.3.1.26) enzyme was successfully demonstrated for the first time; progress of the reaction was followed by spectrophotometric determination at 315 nm. The following conditions were optimized for maximal formation of p-HCAM: pH (8.5), temperature (37°C), speed of agitation (50 rpm), enzyme concentration (0.080 µM), and substrate concentration (0.50 mM). Under these conditions, the yield of the reaction was ∼15% in 1 h incubation period and ∼63% after an overnight (∼18 h) incubation period. The product (p-HCAM) of the reaction of PTAL with L-TM was confirmed using Nuclear Magnetic Resonance spectroscopy (NMR). Fourier Transform Infra-Red spectroscopy (FTIR) was carried out to rule out potential hydrolysis of p-HCAM during overnight incubation. Potential antibacterial activity of p-HCAM was tested against several strains of Gram-positive and Gram-negative bacteria. This study describes a synthetically useful transformation, and could have future clinical and industrial applications.

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium.

The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.

New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models.

Tyrosine ammonia lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid in purple phototropic bacteria and Actinomycetales. The enzyme is used in bioengineering and has the potential to be used industrially. It belongs to a family of enzymes that uses a 4-methylidene-imidazole-5-one (MIO) cofactor to catalyze the deamination amino acids. In the present work, we used a QM/MM and a QM cluster models of TAL to explore two putative reaction paths for its catalytic mechanism. Part of the N-MIO mechanism was previously studied by computational methods. We improved on previous studies by using a larger, more complete model of the enzyme, and by describing the complete reaction path. The activation energy for this mechanism, in agreement with the previous study, is 28.5 kcal/mol. We also found another reaction path that has overall better kinetics and reaches the products in a single reaction step. The barrier for this Single-Step mechanism is 16.6 kcal/mol, which agrees very well with the experimental kcat of 16.0 kcal/mol. The geometrical parameters obtained for the cluster and QM/MM models are very similar, despite differences in the relative energies. This means that both approaches are capable of describing the correct catalytic path of TAL.