Metabolic Engineering of Escherichia coli for High-Level Production of l-Phenylalanine.

l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.

Comparing trophic position estimates using bulk and compound specific stable isotope analyses: applying new approaches to mackerel icefish Champsocephalus gunnari.

Quantifying the tropic position (TP) of an animal species is key to understanding its ecosystem function. While both bulk and compound-specific analyses of stable isotopes are widely used for this purpose, few studies have assessed the consistency between and within such approaches. Champsocephalus gunnari is a specialist teleost that predates almost exclusively on Antarctic krill Euphausia superba. This well-known and nearly constant trophic relationship makes C. gunnari particularly suitable for assessing consistency between TP methods under field conditions. In the present work, we produced and compared TP estimates for C. gunnari and its main prey using a standard bulk and two amino acid-specific stable isotope approaches (CSI-AA). One based on the difference between glutamate and phenylalanine (TPGlx-Phe), and the other on the proline-phenylalanine difference (TPPro-Phe). To do that, samples from C. gunnari, E. superba and four other pelagic invertebrate and fish species, all potential prey for C.gunnari, were collected off the South Orkney Islands between January and March 2019, analyzed using standard isotopic ratio mass spectrometry methods and interpreted following a Bayesian approach. Median estimates (CI95%) for C. gunnari were similar between TPbulk (3.6; CI95%: 3.0-4.8) and TPGlx-Phe(3.4; CI95%:3.2-3.6), and lower for TPPro-Phe (3.1; CI95%:3.0-3.3). TP differences between C. gunnari and E. superba were 1.4, 1.1 and 1.2, all compatible with expectations from the monospecific diet of this predator (ΔTP=1). While these results suggest greater accuracy for Glx-Phe and Pro-Phe, differences observed between both CSI-AA approaches suggests these methods may require further validation before becoming a standard tool for trophic ecology.

Impact of distinct FG nucleoporin repeats on Nup98 self-association.

Nucleoporins rich in phenylalanine/glycine (FG) residues form the permeability barrier within the nuclear pore complex and are implicated in several pathological cellular processes, including oncogenic fusion condensates. The self-association of FG-repeat proteins and interactions between FG-repeats play a critical role in these activities by forming hydrogel-like structures. Here we show that mutation of specific FG repeats of Nup98 can strongly decrease the protein's self-association capabilities. We further present a cryo-electron microscopy structure of a Nup98 peptide fibril with higher stability per residue compared with previous Nup98 fibril structures. The high-resolution structure reveals zipper-like hydrophobic patches which contain a GLFG motif and are less compatible for binding to nuclear transport receptors. The identified distinct molecular properties of different regions of the nucleoporin may contribute to spatial variations in the self-association of FG-repeats, potentially influencing transport processes through the nuclear pore.

Laboratory parameters related to disease severity and physical performance after reconvalescence of acute COVID-19 infection.

Research into the molecular basis of disease trajectory and Long-COVID is important to get insights toward underlying pathophysiological processes. The objective of this study was to investigate inflammation-mediated changes of metabolism in patients with acute COVID-19 infection and throughout a one-year follow up period. The study enrolled 34 patients with moderate to severe COVID-19 infection admitted to the University Clinic of Innsbruck in early 2020. The dynamics of multiple laboratory parameters (including inflammatory markers [C-reactive protein (CRP), interleukin-6 (IL-6), neopterin] as well as amino acids [tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr)], and parameters of iron and vitamin B metabolism) was related to disease severity and patients' physical performance. Also, symptom load during acute illness and at approximately 60 days (FU1), and one year after symptom onset (FU2) were monitored and related with changes of the investigated laboratory parameters: During acute infection many investigated laboratory parameters were elevated (e.g., inflammatory markers, ferritin, kynurenine, phenylalanine) and enhanced tryptophan catabolism and phenylalanine accumulation were found. At FU2 nearly all laboratory markers had declined back to reference ranges. However, kynurenine/tryptophan ratio (Kyn/Trp) and the phenylalanine/tyrosine ratio (Phe/Tyr) were still exceeding the 95th percentile of healthy controls in about two thirds of our cohort at FU2. Lower tryptophan concentrations were associated with B vitamin availability (during acute infection and at FU1), patients with lower vitamin B12 levels at FU1 had a prolonged and more severe impairment of their physical functioning ability. Patients who had fully recovered (ECOG 0) presented with higher concentrations of iron parameters (ferritin, hepcidin, transferrin) and amino acids (phenylalanine, tyrosine) at FU2 compared to patients with restricted ability to work. Persistent symptoms at FU2 were tendentially associated with IFN-γ related parameters. Women were affected by long-term symptoms more frequently. Conclusively, inflammation-mediated biochemical changes appear to be related to symptoms of patients with acute and Long Covid.

Biochemical Detection of Capsid in the Nucleus During HIV-1 Infection.

Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1NL4-3, with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers are also performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions.

[Changes in protein requirements of elderly people over 5 years assessed using the indicator amino acid oxidation method].

OBJECTIVE: To evaluate the changes in protein requirements of the elderly during the past five years. METHODS: Based on the previous study of protein requirements of 14 elderly in 2017, 4 of these elderly(70-80 y) were included as study participants and protein requirements were re-evaluated using the indicator amino acid oxidation method. There were seven protein levels: 0.1, 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 g/(kg·d). Maintenance diets were given for the first two days of each protein level. A stable isotope study was conducted on the day 3, using L-~(13)C-phenylalanine as an indicator on the basis of an amino acid rationed diet, which was orally ingested into the body along with the amino acid rationed diet, and breath and urine samples were collected when the metabolism of L-~(13)C-phenylalanine reached steady state in the body. By measuring the kinetic parameters of labeled amino acids in the samples, a nonlinear mixed-effects model was constructed for the protein intake to be tested and the oxidation rate of labeled amino acids. The mean protein requirement of the study population was determined by the protein intake corresponding to the inflection point of the curve. RESULTS: Based on the production rate of ~(13)CO_2 in exhaled breath of four elderly people at different protein levels, the mean protein requirement was 1.05(95%CI 0.51-1.60) g/(kg·d). The protein recommended nutrient intake was 1.31(95%CI 0.64-2.00) g/(kg·d) was estimated by applying the coefficient of variation of the mean protein requirement to derive the recommended nutrient intake. CONCLUSION: Protein requirements in the elderly have increased over a five-year period and sarcopenia may be the main cause of increased protein requirements.

Expression and purification of fluorinated proteins from mammalian suspension culture.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Traditional medicinal use is linked with apparency, not specialized metabolite profiles in the order Caryophyllales.

PREMISE: Better understanding of the relationship between plant specialized metabolism and traditional medicine has the potential to aid in bioprospecting and untangling of cross-cultural use patterns. However, given the limited information available for metabolites in most plant species, understanding medicinal use-metabolite relationships can be difficult. The order Caryophyllales has a unique pattern of lineages of tyrosine- or phenylalanine-dominated specialized metabolism, represented by mutually exclusive anthocyanin and betalain pigments, making Caryophyllales a compelling system to explore the relationship between medicine and metabolites by using pigment as a proxy for dominant metabolism. METHODS: We compiled a list of medicinal species in select tyrosine- or phenylalanine-dominant families of Caryophyllales (Nepenthaceae, Polygonaceae, Simmondsiaceae, Microteaceae, Caryophyllaceae, Amaranthaceae, Limeaceae, Molluginaceae, Portulacaceae, Cactaceae, and Nyctaginaceae) by searching scientific literature until no new uses were recovered. We then tested for phylogenetic clustering of uses using a "hot nodes" approach. To test potential non-metabolite drivers of medicinal use, like how often humans encounter a species (apparency), we repeated the analysis using only North American species across the entire order and performed phylogenetic generalized least squares regression (PGLS) with occurrence data from the Global Biodiversity Information Facility (GBIF). RESULTS: We hypothesized families with tyrosine-enriched metabolism would show clustering of different types of medicinal use compared to phenylalanine-enriched metabolism. Instead, wide-ranging, apparent clades in Polygonaceae and Amaranthaceae are overrepresented across nearly all types of medicinal use. CONCLUSIONS: Our results suggest that apparency is a better predictor of medicinal use than metabolism, although metabolism type may still be a contributing factor.

Exogenous 5-Aminolevulinic acid improved low-temperature tolerance tomato seedling by regulating starch content and phenylalanine metabolism.

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL, SlPOD1, and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism.

Discovery of low-molecular-weight phenylalanine derivatives as novel HIV capsid modulators with improved antiretroviral activity and metabolic stability.

The HIV capsid (CA) protein is a promising target for anti-AIDS treatment due to its critical involvement in viral replication. Herein, we utilized the well-documented CA inhibitor PF74 as our lead compound and designed a series of low-molecular-weight phenylalanine derivatives. Among them, compound 7t exhibited remarkable antiviral activity with a high selection index (EC50 = 0.040 µM, SI = 2815), surpassing that of PF74 (EC50 = 0.50 µM, SI = 258). Furthermore, when evaluated against the HIV-2 strain, 7t (EC50 = 0.13 µM) demonstrated approximately 14-fold higher potency than that of PF74 (EC50 = 1.76 µM). Insights obtained from surface plasmon resonance (SPR) revealed that 7t exhibited stronger target affinity to the CA hexamer and monomer in comparison to PF74. The potential interactions between 7t and the HIV-1 CA were further elucidated using molecular docking and molecular dynamics simulations, providing a plausible explanation for the enhanced target affinity with 7t over PF74. Moreover, the metabolic stability assay demonstrated that 7t (T1/2 = 77.0 min) significantly outperforms PF74 (T1/2 = 0.7 min) in human liver microsome, exhibiting an improvement factor of 110-fold. In conclusion, 7t emerges as a promising drug candidate warranting further investigation.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Supplementation for Performance and Health in Patients with Phenylketonuria: An Exercise-Based Approach to Improving Dietary Adherence.

Supplementation is crucial for improving performance and health in phenylketonuria (PKU) patients, who face dietary challenges. Proteins are vital for athletes, supporting muscle growth, minimizing catabolism, and aiding muscle repair and glycogen replenishment post-exercise. However, PKU individuals must limit phenylalanine (Phe) intake, requiring supplementation with Phe-free amino acids or glycomacropeptides. Tailored to meet nutritional needs, these substitutes lack Phe but fulfill protein requirements. Due to limited supplement availability, athletes with PKU may need higher protein intake. Various factors affect tolerated Phe levels, including supplement quantity and age. Adhering to supplement regimens optimizes performance and addresses PKU challenges. Strategically-timed protein substitutes can safely enhance muscle synthesis and sports performance. Individualized intake is essential for optimal outcomes, recognizing proteins' multifaceted role. Here, we explore protein substitute supplementation in PKU patients within the context of physical activity, considering limited evidence.

An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis.

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of 19F nuclei, 19F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid L-4-trifluoromethylphenylalanine (tfmF) was site-specifically introduced into the mitogen-activated protein kinase MEKK3 in V. natriegens using genetically encoded technology, which will be an extensive method for in-cell protein structure and dynamic investigation.

Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

Metformin and feeding increase levels of the appetite-suppressing metabolite Lac-Phe in humans.

Metformin, a widely used first-line treatment for type 2 diabetes (T2D), is known to reduce blood glucose levels and suppress appetite. Here we report a significant elevation of the appetite-suppressing metabolite N-lactoyl phenylalanine (Lac-Phe) in the blood of individuals treated with metformin across seven observational and interventional studies. Furthermore, Lac-Phe levels were found to rise in response to acute metformin administration and post-prandially in patients with T2D or in metabolically healthy volunteers.

Lac-Phe mediates the effects of metformin on food intake and body weight.

Metformin is a widely prescribed anti-diabetic medicine that also reduces body weight. There is ongoing debate about the mechanisms that mediate metformin's effects on energy balance. Here, we show that metformin is a powerful pharmacological inducer of the anorexigenic metabolite N-lactoyl-phenylalanine (Lac-Phe) in cells, in mice and two independent human cohorts. Metformin drives Lac-Phe biosynthesis through the inhibition of complex I, increased glycolytic flux and intracellular lactate mass action. Intestinal epithelial CNDP2+ cells, not macrophages, are the principal in vivo source of basal and metformin-inducible Lac-Phe. Genetic ablation of Lac-Phe biosynthesis in male mice renders animals resistant to the effects of metformin on food intake and body weight. Lastly, mediation analyses support a role for Lac-Phe as a downstream effector of metformin's effects on body mass index in participants of a large population-based observational cohort, the Multi-Ethnic Study of Atherosclerosis. Together, these data establish Lac-Phe as a critical mediator of the body weight-lowering effects of metformin.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering.

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Metabolic engineering of Synechocystis sp. PCC 6803 for the improved production of phenylpropanoids.

BACKGROUND: Phenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis. RESULTS: We eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout. CONCLUSIONS: HPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.