Amino-l
Pilato Hull
The non-proteinogenic aromatic amino acid, p-amino-l-phenylalanine (l-PAPA) is a high-value product with a broad field of applications. In nature, l-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved l-PAPA producing strain.
The general aromatic biosynthesis pathway (also called shikimate pathway) in most microorganisms and plants starts by condensation of the precursors phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) to provide the first intermediate, 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). Eventually (after introduction of another PEP molecule), the last common metabolite, chorismate [1] is formed. From chorismate, various biosynthetic pathways diverge which give rise to a plethora of aromatic compounds. Among them are three proteinogenic aromatic amino acids [l-phenylalanine (l-Phe), l-tyrosine (l-Tyr), and l-tryptophan (l-Trp)] and aromatic vitamins such as quinones, folate (via p-aminobenzoate), and p-hydroxybenzoate [2,3,4,5]. Quinones (ubiquinone, menaquinone, plastoquinone) fulfill important roles in the electron transport chain [3, 6]. Enterobactin (from 2,3-dihydroxybenzoate) is used as an iron complexing siderophore in E. coli, folate is involved in C1 metabolism, and vitamin E in photosynthetic organisms is involved in protection against oxidative stress [7,8,9]. Various secondary metabolites such as phenylpropanoids (mainly in plants) and some antibiotics (chloramphenicol, pristinamycin, and others) in streptomycetes are as well derived from the aromatic pathway [10,11,12].
The rare, non-proteinogenic aromatic amino acid, para-amino-l-phenylalanine (l-PAPA) is used for technical and pharmaceutical applications [22,23,24,25] and has been described as a building block of the anticancer drug, Melphalan [26, 27]. In nature, l-PAPA occurs in plant seeds (Vigna vexillata; [28]), and is an intermediate of the chloramphenicol and pristinamycin biosynthesis pathways in Streptomyces venezuelae and S. pristinaespiralis [29,30,31]. l-PAPA is also a precursor of the antibiotic obafluorin -lactone of Pseudomonas fluorescens [32] and of GameXPeptides in the entomopathogenic bacterium, Photorhabdus luminescens [33]. l-PAPA was moreover successfully used as precursor in the biosynthetic diversification of jadomycin production with S. venezuelae cells [34]. L-PAPA has also been used for the synthesis of the biopolyamide precursor, 4-aminohydrocinnamic acid [35, 36].
A preferred carbon source for the production of aromatic amino acids with E. coli is glucose. Glycerol which can be used as an alternative carbon and energy source by E. coli, is especially attractive for the production of aromatic amino acids [15, 18, 48]. Glycerol can be taken up in E. coli either via the glycerol facilitator, GlpF or by unassisted diffusion [49]. After phosphorylation by the ATP-dependent glycerol kinase (GlpK; [50]), and an oxidation step by glycerol 3-phosphate dehydrogenase (GlpD; [51]) dihydroxyacetone phosphate (DHAP) can enter the lower glycolysis. Glycerol has no competing application in the food industry and it appears as a byproduct during biodiesel production [52]. Here we describe the construction of E. coli strains for the production of valuable l-PAPA using glycerol as sole carbon and energy source.
Expansion of the chorismate pathway of E. coli to produce a variety of non-standard aromatic compounds has already been very successful in many cases. Pioneering work has been done by the group of Frost and others already in the 1990s [61,62,63,64, 66]. Thereafter, processes have been published for p-hydroxybenzoic acid [61, 65], p-aminobenzoic acid [40, 42], protocatechuic acid and catechol [65], vanillin [67, 68], anthranilic acid [69], δ-tocotrienol (a vitamin E compound, [70]), phenol [71,72,73,74], salicylic acid and muconic acid [5, 75], styrene, cinnamic acid and hydroxylated derivatives thereof [7, 76,77,78,79,80], tyrosol [81], (hydroxyl)phenyllactic acid, tyramine and other l-Phe- or l-Tyr-derived compounds [76], rosmarinic acid and flavonoids [82, 83], antitumor drugs like violacein and deoxyviolacein [84], several plant alkaloids [85], or even opiates like thebaine, reticuline or hydrocodone [86]. Further examples may be found in recent review articles [79, 87, 88].
To further improve the E4P precursor supply it can be considered to further increase the number of gene copies in the E. coli genome as only one additional copy of tktA and glpX was inserted. The enhanced flux through the shikimate pathway by overexpression of plasmid-borne aroFBL genes showed a beneficial effect in our study. Other studies have shown that the overexpression of the other shikimate pathway genes (aroA, C, D and E) as well improved the formation of l-Tyr which is derived from chorismate [14]. It is likely that a positive effect can be also observed for the l-PAPA production. This improved route to l-PAPA based upon a simple medium with a sustainable carbon source which is currently in ample supply from biodiesel production and which does not compete with human nutrition, opens up the possibility to produce an interesting building block in larger scale.
This study demonstrated that E. coli is a good chassis strain for l-PAPA production using glycerol as an alternative sole carbon source. We constructed by metabolic grafting a de novo pathway for l-PAPA in E. coli. By improving the E4P precursor supply and the increased flux through the shikimate pathway both l-PAPA titer and yield were augmented.
Chemicals, antibiotics, buffer components, culture media and analytical standards, used in this study were purchased from AppliChem GmbH (Darmstadt, Germany), Carl Roth GmbH (Karlsruhe, Germany), or Sigma-Aldrich/Fluka (Taufkirchen, Germany) and were of the highest available purity. l-PAPA was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).
The deletion of the gene tyrR, encoding the regulator of the tyr regulon, was carried out according to a λred-recombineering method [55]. A linear DNA fragment containing the FRT-flanked chloramphenicol resistance (cat) cassette was amplified from plasmid pCAS30-FRT-cat-FRT [57] using the primer pair DeltyrRFw/-Rw (see Table 1 for sequences). The thus obtained amplified linear DNA was introduced by electroporation into electrocompetent E. coli FUS4.7 cells that carried the Red recombinase expression vector pKD46 [55]. After confirmation of the FRT-cat-FRT integration by colony PCR with the primer pair Ko-tyrRFw/-Rw (see Table 1), the cat marker was removed by transient expression of a FLP recombinase from plasmid pCP20 [56] to eventually generate the tyrR deletion strain, FUS4.7R. Verification of the disruption was performed using colony PCR with primers up- and downstream of disrupted regions (Ko-tyrR-Fw/-Rw). Finally, cells were grown at 42 C to remove the temperature-sensitive replicons, pCP20 or pKD46, respectively [55].
The authors are grateful to Dr. Josef Altenbuchner (Institute of Industrial Genetics, University of Stuttgart) for providing the plasmid pJeM2 and to Dr. Bernd A. Nebel (Institute of Technical Biochemistry, University of Stuttgart) for helping with the MS measurements. We thank Ursula Degner (Research Center Jlich, Germany) for help with the cloning work leading to plasmids pF-109 and pF-112 and Alexander Dietrich (Institute of Biochemical Engineering, University of Stuttgart) for technical assistance with the bioreactor. We thank the Ministerium fr Wissenschaft und Kunst Baden-Wuerttemberg for financial support.
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High-molecular-weight polypeptides with functional aromatic side chains, poly(4-amino-l-phenylalanine), were prepared by the metal-initiated polymerization of theNα-carboxyanhydride of the corresponding amino acid, which is a microbial derivative of phenylalanine.
High-molecular-weight polypeptides with functional aromatic side chains, poly(4-amino-l-phenylalanine), were prepared by the metal-initiated polymerization of theN\u03B1-carboxyanhydride of the corresponding amino acid, which is a microbial derivative of phenylalanine.
AbstractThe reactivity of trifluoromethanesulfonyl esters derived from l-lyxofuranosides and l-lyxopyranosides was investigated with various 5-aminopyrimidines as nucleophiles with the expectation to synthesize N-substituted 5-amino-ribosugars. The lyxopyranoside forms were found to be unreactive, while the lyxofuranoside forms were found to be reactive with 5-aminopyrimidines, yielding novel N-substituted 5-amino-lyxofuranosides. We report on the synthesis of these novel N-substituted lyxofuranosides and the systematic analyses of NMR data that demonstrate trends within each series: furano-, pyrano-, β- and α- anomers of l-lyxose and β-d-ribopyranoside forms. The data call for caution when identifying these monosaccharides in isomeric mixtures.
By employing chip-based capillary zone electrophoresis coupled to high-resolution mass spectrometry, we profiled the plasma metabolome of 134 patients diagnosed with sporadic amyotrophic lateral sclerosis (ALS) (81 males and 53 females) and 118 individuals deemed healthy (49 males and 69 females). The most significant markers (p < 0.01) were creatine, which was 49% elevated, and creatinine and methylhistidine, which were decreased by 20 and 24%, respectively, in ALS patients. The ratio of creatine versus creatinine increased 370 and 200% for male and female ALS patients, respectively. In addition, male ALS patients on an average had 5-13% lower amounts of seven essential amino acids, whereas females did not significantly differ from healthy controls. We developed two models using the metabolite abundances: (1) a classification model for the separation of ALS and healthy samples and (2) a classification model for the prediction of disease progression based on the ALS functional rating score. Utilizing a Monte Carlo cross-validation approach, a linear discriminant analysis model achieved a mean area under the receiver operating characteristic curve (AUC) of 0.85 (0.06) with a mean sensitivity of 80% (9%) and specificity of 78% (10%) for the separation of ALS and controls, respectively. A support vector machine classifier predicted progression categories with an AUC of 0.90 (0.06) with a mean sensitivity of 73% (10%) and a specificity of 86% (5%). Lastly, using a previously reported assay with a stable isotope-labeled (13C315N2) spike-in standard, we were unable to detect the exogenous neurotoxic metabolite, β-methylamino-l-alanine, in the free or protein-bound fraction of any of the 252 plasma samples.
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