Accessing p-Hydroxycinnamic Acids: Chemical Synthesis, Biomass Recovery, or Engineered Microbial Production?

p-Hydroxycinnamic acids (i. e., p-coumaric, ferulic, sinapic, and caffeic acids) are phenolic compounds involved in the biosynthesis pathway of lignin. These naturally occurring molecules not only exhibit numerous attractive properties, such as antioxidant, anti-UV, and anticancer activities, but they also have been used as building blocks for the synthesis of tailored monomers and functional additives for the food/feed, cosmetic, and plastics sectors. Despite their numerous high value-added applications, the sourcing of p-hydroxycinnamic acids is not ensured at the industrial scale except for ferulic acid, and their production cost remains too high for commodity applications. These compounds can be either chemically synthesized or extracted from lignocellulosic biomass, and recently their production through bioconversion emerged. Herein the different strategies described in the literature to produce these valuable molecules are discussed.

Neuroprotection of hydroxysafflor yellow A in experimental cerebral ischemia/reperfusion injury via metabolic inhibition of phenylalanine and mitochondrial biogenesis.

Stroke is the second most frequent cause of mortality, resulting in a huge societal burden worldwide. Timely reperfusion is the most effective therapy; however, it is difficult to prevent ischemia/reperfusion (I/R) injury. In traditional Chinese medicine, hydroxysafflor yellow A (HSYA) has been widely used for the treatment of cerebrovascular disease and as a protective therapy against I/R injury. Evidence has demonstrated that HSYA could reduce the levels of reactive oxygen species and suppress cellular apoptosis; however, whether HSYA alters the metabolic profile as its underlying mechanism for neuroprotection remains unknown. In the present study, using a metabolomic screening, phenylalanine was identified to significantly increase in an experimental model of mouse cerebral I/R injury. Notably, western blotting and qPCR analysis were conducted to test the expression level of apoptosis­associated factors, and HSYA was identified to be able to protect neuronal cells by reducing phenylalanine level associated with I/R injury. Additionally, these findings were confirmed in primary mouse neurons and PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) stress. Of note, HSYA was observed to regulate the mRNA expression of key metabolic enzymes, phenylalanine hydroxylase, tyrosine aminotransferase and aspartate aminotransferase, which are responsible for phenylalanine metabolism. Furthermore, by performing mitochondrial labeling and JC­1 fluorescence assay, HSYA was identified to promote mitochondrial function and biogenesis suppressed by OGD/R. The findings of the present study demonstrated that I/R injury could increase the levels of phenylalanine, and HSYA may inhibit phenylalanine synthesis to enhance mitochondrial function and biogenesis for neuroprotection. The present study proposed a novel metabolite biomarker for cerebral I/R injury and the evaluated the efficacy of HSYA as a potential therapeutic treatment I/R injury.

Enhancement of L-phenylalanine production by engineered Escherichia coli using phased exponential L-tyrosine feeding combined with nitrogen source optimization.

Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation.

Insight into the transmission biology and species-specific functional capabilities of tsetse (Diptera: glossinidae) obligate symbiont Wigglesworthia.

UNLABELLED: Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species. IMPORTANCE: Genomic stasis has historically been associated with obligate endosymbionts and their sister species. Here we characterize the Wigglesworthia genome of the tsetse fly species Glossina morsitans and compare it to its sister genome within G. brevipalpis. The similarity and variation between the genomes enabled specific hypotheses regarding functional biology. Expression analyses indicate significant levels of transcriptional regulation and support development- and tissue-specific functional roles for the symbiosis previously not observed in obligate mutualist symbionts. Retention of the genetically expensive flagella within these small genomes was demonstrated to be significant in symbiont transmission and tailored to the unique tsetse fly reproductive biology. Distinctions in metabolomes were also observed. We speculate an additional role for Wigglesworthia symbiosis where infections with pathogenic trypanosomes may depend upon symbiont species-specific metabolic products and thus influence the vector competence traits of different tsetse fly host species.

Characterization of L-phenylalanine metabolism to acetophenone and 1-phenylethanol in the flowers of Camellia sinensis using stable isotope labeling.

Acetophenone (AP) and 1-phenylethanol (1PE) are the two major endogenous volatile compounds in flowers of Camellia sinensis var. Yabukita. Until now no information has been available on the biosynthesis of AP and 1PE in plants. Here we propose that AP and 1PE are derived from L-phenylalanine (L-Phe), based on feeding experiments using stable isotope-labeled precursors L-[(2)H(8)]Phe and L-[(13)C(9)]Phe. The subacid conditions in the flowers result in more hydrogenation than dehydrogenation in the transformation between AP and 1PE. Due to the action of some enzyme(s) responsible for the formation of (R)-1PE from AP in the flowers, (R)-1PE is the dominant endogenous steroisomer of 1PE. The modification of 1PE into nonvolatile glycosidic forms is one of the reasons for why only a little 1PE is released from the flowers. The levels of AP, 1PE, and glycosides of 1PE increase during floral development, whereas the level of L-Phe decreases. These metabolites occur mostly in the anthers.

Metabolomics data exploration guided by prior knowledge.

In metabolomics research, it is often important to focus the data analysis to specific areas of interest within the metabolome. In this paper, we describe the application of consensus principal component analysis (CPCA) and canonical correlation analysis (CCA) as a means to explore the relation between metabolome data and (i) biochemically related metabolites and (ii) an amino acid biosynthesis pathway. CPCA searches for major trends in the behavior of metabolite concentrations that are in common for the metabolites of interest and the remainder of the metabolome. CCA identifies the strongest correlations between the metabolites of interest and the remainder of the metabolome. CPCA and CCA were applied to two different microbial metabolomics data sets. The first data set, derived from Pseudomonas putida S12, was relatively simple as it contained metabolomes obtained under four environmental conditions only. The second data set, obtained from Escherichia coli, was much more complex as it consisted of metabolomes obtained under 28 different environmental conditions. In case of the simple and coherent P. putida S12 data set, CCA and CPCA gave similar results as the variation in the subset of the selected metabolites and the remainder of the metabolome was similar. In contrast, CCA and CPCA yielded different results in case of the E. coli data set. With CPCA the trends in the selected subset--the phenylalanine biosynthesis pathway--dominated the results. The main trends were related to high and low phenylalanine productivity, and the metabolites showing a similar behavior in concentration were metabolites regulating the phenylalanine biosynthesis route in the subset and metabolites related to general amino acid metabolism in the remainder of the metabolome. With CCA, neither subset truly dominated the data analysis. CCA described the differences between the wild type and the overproducing strain and the differences between the succinate and glucose grown cells. For the difference between the wild type and the overproducing strain, metabolites from the beginning and the end of aromatic amino acid pathways like erythrose-4-phosphate, tryptophan, and phenylalanine were important for the selected metabolites. CCA and CPCA proved to be complementary data analysis tools that enable the focusing of the data analysis on groups of metabolites that are of specific interest in relation to the remainder of the metabolome. Compared to an ordinary PCA, focusing the data analysis on biologically relevant metabolites lead especially for the complex E. coli data to a better biological interpretation of the data.

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments.

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.

Impaired arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) acid synthesis by phenylalanine metabolites as etiological factors in the neuropathology of phenylketonuria.

The recent literature on polyunsaturated fatty acid metabolism in phenylketonuria (PKU) is critically analyzed. The data suggest that developmental impairment of the accretion of brain arachidonic (20:4n-6) and docosahexaenoic (22:6n-3, DHA) acids is a major etiological factor in the microcephaly and mental retardation of uncontrolled PKU and maternal PKU. These fatty acids appear to be synthesized by the recently elucidated carnitine-dependent, channeled, mitochondrial fatty acid desaturases for which alpha-tocopherolquinone (alpha-TQ) is an essential enzyme cofactor. alpha-TQ can be synthesized either de novo or from alpha-tocopherol. The fetus and newborn would primarily rely on de novo alpha-TQ synthesis for these mitochondrial desaturases because of low maternal transfer of alpha-tocopherol. Homogentisate, a pivotal intermediate in the de novo pathway of alpha-TQ synthesis, is synthesized by 4-hydroxyphenylpyruvate dioxygenase. The major catabolic products of excess phenylalanine, viz. phenylpyruvate and phenyllactate, are proposed to inhibit alpha-TQ synthesis at the level of the dioxygenase reaction by competing with its 4-hydroxyphenylpyruvate substrate, thus leading to a developmental impairment of 20:4n-6 and 22:6n-3 synthesis in uncontrolled PKU and fetuses of PKU mothers. The data suggest that dietary supplementation with carnitine, 20:4n-6, and 22:6n-3 may have therapeutic value for PKU mothers and for PKU patients who have been shown to have a low plasma status of these essential metabolites.

Characterization of the mouse phenylalanine hydroxylase mutation Pah(enu3).

Phenylketonuria (PKU) is an inborn error of metabolism that is inherited in an autosomal recessive manner. It arises from a deficiency of phenylalanine hydroxylase, which is responsible for converting phenylalanine to tyrosine and thereby hastening its catabolism. To produce mouse models for the study of PKU, male mice were mutagenized with ethylnitrosourea and their progeny were screened for the elevated phenylalanine levels characteristic of phenylalanine hydroxylase deficiency. Of three mutant alleles recovered, two (Pah(enu1) and Pah(enu2)) were characterized previously and shown to be missense mutations. Sequencing of phenylalanine hydroxylase cDNA from the third mutant allele, Pah(enu3), revealed that two differently sized transcripts were being produced. These transcripts contained either a 5-nucleotide insertion or a 5-nucleotide deletion and both of these modifications occurred at the same location, the exon 11-exon 12 junction. Sequencing of the exon 11-intron 11 boundary revealed a T --> G transversion in the invariant GT dinucleotide of the wild-type 5' splice donor site. The analogous human Pah mutation would be called c.1199 + 2T > G. Sequence analysis also revealed two cryptic splice donor sites, upstream and downstream of the wild-type splice site, that appear to be used when the wild type is ablated and to thereby yield the observed differently sized transcripts. The 5-nucleotide insertion and the 5-nucleotide deletion are both predicted to cause frame shifting in exon 12 and exon 13, leading to premature termination.

Amonabactin, a novel tryptophan- or phenylalanine-containing phenolate siderophore in Aeromonas hydrophila.

Aeromonas hydrophila 495A2 excreted two forms of amonabactin, a new phenolate siderophore composed of 2,3-dihydroxybenzoic acid, lysine, glycine, and either tryptophan (amonabactin T) or phenylalanine (amonabactin P). Supplementing cultures with L-tryptophan (0.3 mM) caused exclusive synthesis of amonabactin T, whereas supplements of L-phenylalanine (0.3 to 30 mM) gave predominant production of amonabactin P. The two forms of amonabactin were separately purified by a combination of production and polyamide column chromatographic methods. Both forms were biologically active, stimulating growth in iron-deficient medium of an amonabactin-negative mutant. Of 43 additional siderophore-producing isolates of the Aeromonas species that were tested, 76% (19 of 25) of the A. hydrophila isolates were amonabactin positive, whereas only 19% (3 of 16) of the A. sobria isolates and all (3 of 3) of the A. caviae isolates produced amonabactin, suggesting a predominant synthesis of amonabactin in certain Aeromonas species.

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine.

1. Mutants derived from the hydrogen bacterium Alcaligenes eutrophus strain H 16 auxotrophic for phenylalanine and tyrosine were isolated employing mutagenic agents (EMS, nitrite), the colistine counterselection technique and the "pin-point" isolation method. Three different types of mutants were found: (1) Mutants, requiring phenylalanine or phenylpyruvate for growth, were affected in chorismate mutase as well as prephenate dehydratase. Both activities were regained by reversion to prototrophy. The auxotrophic strains accumulated chorismic acid. (2) Strains with a growth response similar to that of the first group lacked only prephenate dehydratase activity which was partially regained by reversion. Chorismate mutase and prephenate dehydrogenase were derepressed up to two-fold. Mutants grown in minimal medium excreted prephenic acid. (3) The third type of mutants required phenylalanine or phenylpyruvate and grew slowly when supplemented with chorismate or prephenate. The enzymes involved in the specific pathway of phenylalanine and tyrosine were found to be present. Some of them were even more active than in the wild-type. 2. Mutants accumulating chorismic acid or prepheric acid were able to grow on minimal medium when incubated long enough. The chemical instability of the excretion products resulted in their nonenzymatic conversion to subsequent intermediates which were taken up by the cells, allowing growth. 3. A method is described for preparing barium prephenate using the auxotrophic mutant 6B-1 derived from A.eutrophus H 16. Prephenic acid, excreted by this strain, was obtained from the culture filtrate with a purity of at least 70% and a yield of approximately 180 mg per 21 of medium.