Production Of L-phenylalanine Research Articles

Effects of structure similar additive on the crystallization of L-phenylalanine from aqueous solution

A comprehensive understanding of the effect of structure similar additives on the nucleation and growth of amino acid in aqueous solution is of great significance for the crystallization of L-phenylalanine and industrial production of anhydrous L-phenylalanine. This paper mainly focuses on the influence of L-tyrosine on the crystallization process of L-phenylalanine by investigating the variation and typical phenomenon in solubility, metastable zone width, crystal nucleation and growth process, and crystal morphology. The results show that the existence of L-tyrosine can obviously increase the solubility and narrow the metastable zone of L-phenylalanine thus promote the nucleation process. Meanwhile, L-tyrosine can also selectively inhibit the formation of monohydrate and improve the purity of industry-favored anhydrous L-phenylalanine in the final product. In order to explore the inhibiting effect of L-tyrosine in crystallization process, the molecular configurations in two different crystallization states were simulated by using Molecular Dynamics.The results show that water and L-phenylalanine molecules tend to form anhydrous crystals when the addition proportion of L-tyrosine increases. The theoretical calculations are in good agreement with the experimental data. The conclusions of this paper will provide new ideas and effective controlling approaches for L-phenylalanine crystals’ morphology and amino acids’ crystallization process.

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.

Biosynthesis of fragrance 2-phenylethanol from sugars by Pseudomonasputida

BackgroundPetrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries offer eco-friendly alternatives. The solvent-tolerant Pseudomonasputida DOT-T1E serves as a suitable host for producing aromatic compounds, specifically l-phenylalanine and its derivative, 2-phenylethanol (2-PE), which find widespread applications in various industries.ResultsThis study focuses on enhancing 2-PE production in two l-phenylalanine overproducing strains of DOT-T1E, namely CM12-5 and CM12-5Δgcd (xylABE), which grow with glucose and glucose-xylose, respectively. To synthesize 2-PE from l-phenylalanine, these strains were transformed with plasmid pPE-1, bearing the Ehrlich pathway genes, and it was found higher 2-PE production with glucose (about 50–60 ppm) than with xylose (< 3 ppm). To understand the limiting factors, we tested the addition of phenylalanine and intermediates from the Ehrlich and shikimate pathways. The results identified intracellular l-phenylalanine as a key limiting factor for 2-PE production. To overcame this limitation, a chorismate mutase/prephenate dehydratase variant—insentive to feedback inhibition by aromatic amino acids—was introduced in the producing strains. This led to increased l-phenylalanine production and subsequently produced more 2-PE (100 ppm). Random mutagenesis of the strains also produced strains with higher l-phenylalanine titers and increased 2-PE production (up to 120 ppm). The improvements resulted from preventing dead-end product accumulation from shikimate and limiting the catabolism of potential pathway intermediates in the Ehrlich pathway. The study explored agricultural waste substrates, such as corn stover, sugarcane straw and corn-syrup as potential C sources. The best results were obtained using 2G substrates at 3% (between 82 and 100 ppm 2-PE), with glucose being the preferred sugar for 2-PE production among the monomeric sugars in these substrates.ConclusionsThe findings of this study offer strategies to enhance phenylalanine production, a key substrate for the synthesis of aromatic compounds. The ability of P.putida DOT-T1E to thrive with various C-sources and its tolerance to substrates, products, and potential toxicants in industrial wastes, are highlighted. The study identified and overcome possible bottlenecks for 2-PE production. Ultimately, the strains have potential to become efficient microbial platforms for synthesizing 2-PE from agro-industrial waste materials.

Systematic engineering enables efficient biosynthesis of L-phenylalanine in E. coli from inexpensive aromatic precursors

BackgroundL-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant.ResultsThe engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate.ConclusionSystematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Construction of Recombinant Escherichia coli with a High L-Phenylalanine Production Yield from Glucose

L-phenylalanine is an important aromatic amino acid that is widely used in the area of feed, food additives, and pharmaceuticals. Among the different strategies of L-phenylalanine synthesis, direct microbial fermentation from raw substrates has attracted more and more attention due to its environment friendly process and low-cost raw materials. In this study, a rational designed recombinant Escherichia coli was constructed for L-phenylalanine production. Based on wild type E. coli MG1655, multilevel engineering strategies were carried out, such as directing more carbon flux into the L-phenylalanine synthetic pathway, increasing intracellular level of precursors, blocking by-product synthesis pathways and facilitating the secretion of L-phenylalanine. During 5 L fed batch fermentation, recombinant E. coli MPH-3 could produce 19.24 g/L of L-phenylalanine with a yield of 0.279 g/g glucose. To the best of our knowledge, this is one of the highest yields of L-phenylalanine producing E. coli using glucose as the sole carbon source in fed-batch fermentation.

Impact of mixing insufficiencies on L-phenylalanine production with an Escherichia coli reporter strain in a novel two-compartment bioreactor

BackgroundThe omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood.ResultsTherefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L−1 demonstrating the consistency of this study’s microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process.ConclusionsThe here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.

Transcriptomics and metabolomics analysis of L-phenylalanine overproduction in Escherichia coli

BackgroundHighly efficient production of L-phenylalanine (L-Phe) in E. coli has been achieved by multiple rounds of random mutagenesis and modification of key genes of the shikimate (SHIK) and L-Phe branch pathways. In this study, we performed transcriptomic (16, 24 and 48 h) and metabolomic analyses (8, 16, 24, 32,40, and 48 h) based on time sequences in an engineered E. coli strain producing L-Phe, aiming to reveal the overall changes of metabolic activities during the fermentation process.ResultsThe largest biomass increase rate and the highest production rate were seen at 16 h and 24 h of fermentation, respectively reaching 5.9 h−1 and 2.76 g/L/h, while the maximal L-Phe titer of 60 g/L was accumulated after 48 h of fermentation. The DEGs and metabolites involved in the EMP, PP, TCA, SHIIK and L-Phe-branch pathways showed significant differences at different stages of fermentation. Specifically, the significant upregulation of genes encoding rate-limiting enzymes (aroD and yidB) and key genes (aroF, pheA and aspC) pushed more carbon flux toward the L-Phe synthesis. The RIA changes of a number of important metabolites (DAHP, DHS, DHQ, Glu and PPN) enabled the adequate supply of precursors for high-yield L-Phe production. In addition, other genes related to Glc transport and phosphate metabolism increased the absorption of Glc and contributed to rerouting the carbon flux into the L-Phe-branch.ConclusionsTranscriptomic and metabolomic analyses of an L-Phe overproducing strain of E. coli confirmed that precursor supply was not a major limiting factor in this strain, whereas the rational distribution of metabolic fluxes was achieved by redistributing the carbon flux (for example, the expression intensity of the genes tyrB, aspC, aroL and aroF/G/H or the activity of these enzymes is increased to some extent), which is the optimal strategy for enhancing L-Phe production.

Shikimate Kinase Plays Important Roles in Anthocyanin Synthesis in Petunia.

In plants, the shikimate pathway is responsible for the production of aromatic amino acids L-tryptophan, L-phenylalanine, and L-tyrosine. L-Phenylalanine is the upstream substrate of flavonoid and anthocyanin synthesis. Shikimate kinase (SK) catalyzes the phosphorylation of the C3 hydroxyl group of shikimate to produce 3-phosphate shikimate (S3P), the fifth step of the shikimate pathway. However, whether SK participates in flavonoid and anthocyanin synthesis is unknown. This study characterized the single-copy PhSK gene in the petunia (Petunia hybrida) genome. PhSK was localized in chloroplasts. PhSK showed a high transcription level in corollas, especially in the coloring stage of flower buds. Suppression of PhSK changed flower color and shape, reduced the content of anthocyanins, and changed the flavonoid metabolome profile in petunia. Surprisingly, PhSK silencing caused a reduction in the shikimate, a substrate of PhSK. Further qPCR analysis showed that PhSK silencing resulted in a reduction in the mRNA level of PhDHQ/SDH, which encodes the protein catalyzing the third and fourth steps of the shikimate pathway, showing a feedback regulation mechanism of gene expression in the shikimate pathway.

Engineering of global transcription factor FruR to redirect the carbon flow in Escherichia coli for enhancing l-phenylalanine biosynthesis

BackgroundThe catabolite repressor/activator protein (FruR) is a global regulatory protein known to control the expression of several genes concerned with carbon utilization and energy metabolism. This study aimed to illustrate effects of the FruR mutant on the L-phenylalanine (L-PHE) producing strain PHE01.ResultsRandom mutagenesis libraries of fruR generated in vitro were first integrated into the chromosome of PHE01 by CRISPR/Cas9 technique, and then the best mutant PHE07 (FruRE173K) was obtained. With this mutant, a final L-PHE concentration of 70.50 ± 1.02 g/L was achieved, which was 23.34% higher than that of PHE01. To better understand the mechanism, both transcriptomes and metabolomes of PHE07 were carried out and compared to that of PHE01. Specifically, the transcript levels of genes involved in gluconeogenesis pathway, pentose phosphate pathway, Krebs cycle, and glyoxylate shunt were up-regulated in the FruRE173K mutant, whereas genes aceEF, acnB, and icd were down-regulated. From the metabolite level, the FruRE173K mutation led to an accumulation of pentose phosphate pathway and Krebs cycle products, whereas the products of pyruvate metabolism pathway: acetyl-CoA and cis-aconic acid, were down-regulated. As a result of the altered metabolic flows, the utilization of carbon sources was improved and the supply of precursors (phosphoenolpyruvate and erythrose 4-phosphate) for L-PHE biosynthesis was increased, which together led to the enhanced production of L-PHE.ConclusionA novel strategy for L-PHE overproduction by modification of the global transcription factor FruR in E. coli was reported. Especially, these findings expand the scope of pathways affected by the fruR regulon and illustrate its importance as a global regulator in L-PHE production.

Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production.

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.

Construction of recombinant Escherichia coli for production of L-phenylalanine-derived compounds.

L-phenylalanine is an important amino acid that is widely used in the fields of food flavors and pharmaceuticals. Apart from L-phenylalanine itself, various commercially valuable chemical compounds can also be generated via the L-phenylalanine biosynthesis pathway. Compared with direct extraction from plants or synthesis by chemical reaction, microbial production of L-phenylalanine -derived compounds can overcome the drawbacks of environmental pollution, low yield, and mixtures of stereoisomeric products. Accordingly, increasing intracellular levels of precursors, deregulating feedback inhibition and transcription repression, engineering global regulators and other effective strategies have been implemented to produce different L-phenylalanine -derived compounds in the excellent chassis host Escherichia coli. Finally, this review highlights principal strategies for improving the production of L-phenylalanine and/or its derivatives in E. coli, and discusses the future outlook for further enhancing the titer and yields of these compounds.

Benzoic acid production via cascade biotransformation and coupled fermentation-biotransformation.

As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L-phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133-146 mM benzoic acid (16.2-17.8 g/Laq ) with 88-97% conversion via seven- and nine-step cascade biotransformation of styrene and L-phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L-phenylalanine with in situ biotransformation to give 63-70 mM benzoic acid (7.6-8.6 g/Laq ), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations.

Application of Dynamic Regulation to Increase L-Phenylalanine Production in Escherichia coli.

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce L-phenylalanine (LPhe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36- fold of the strain xllp1 (45.0 g/l). Moreover, the L-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/ g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.

Metabolic engineering for the production of l-phenylalanine in Escherichia coli.

As one of the three proteinogenic aromatic amino acids, l-phenylalanine is widely applied in the food, chemical and pharmaceutical industries, especially in production of the low-calorie sweetener aspartame. Microbial production of l-phenylalanine has become attractive as it possesses the advantages of environmental friendliness, low cost, and feedstock renewability. With the progress of metabolic engineering, systems biology and synthetic biology, production of l-phenylalanine from glucose in Escherichia coli with relatively high titer has been achieved by improving the intracellular levels of precursors, alleviating transcriptional repression and feedback inhibition of key enzymes, increasing the export of l-phenylalanine, engineering of global regulators, and overexpression of rate-limiting enzymes. In this review, successful metabolic engineering strategies for increasing l-phenylalanine accumulation from glucose in E. coli are described. In addition, perspectives for further improvement of production of l-phenylalanine are discussed.

Plasmid Stability and L-Phenylalanine Production in Escherichia Coli

Plasmid Stability and L-Phenylalanine Production in Escherichia Coli

Biocompatibility and anticancer activity of L-phenyl alanine-coated iron oxide magnetic nanoparticles as potential chrysin delivery system

Abstract

Genetic engineering of Escherichia coli to improve L-phenylalanine production

BackgroundL-phenylalanine (L-Phe) is an essential amino acid for mammals and applications expand into human health and nutritional products. In this study, a system level engineering was conducted to enhance L-Phe biosynthesis in Escherichia coli.ResultsWe inactivated the PTS system and recruited glucose uptake via combinatorial modulation of galP and glk to increase PEP supply in the Xllp01 strain. In addition, the HTH domain of the transcription factor TyrR was engineered to decrease the repression on the transcriptional levels of L-Phe pathway enzymes. Finally, proteomics analysis demonstrated the third step of the SHIK pathway (catalyzed via AroD) as the rate-limiting step for L-Phe production. After optimization of the aroD promoter strength, the titer of L-Phe increased by 13.3%. Analysis of the transcriptional level of genes involved in the central metabolic pathways and L-Phe biosynthesis via RT-PCR showed that the recombinant L-Phe producer exhibited a great capability in the glucose utilization and precursor (PEP and E4P) generation. Via systems level engineering, the L-Phe titer of Xllp21 strain reached 72.9 g/L in a 5 L fermenter under the non-optimized fermentation conditions, which was 1.62-times that of the original strain Xllp01.ConclusionThe metabolic engineering strategy reported here can be broadly employed for developing genetically defined organisms for the efficient production of other aromatic amino acids and derived compounds.

Phosphoenolpyruvate Transporter Enables Targeted Perturbation During Metabolic Analysis of L-Phenylalanine Production With Escherichia coli.

Usually perturbation of the metabolism of cells by addition of substrates is applied for metabolic analysis of production organisms, but perturbation studies are restricted to the endogenous substrates of the cells under study. The goal of this study is to overcome this limitation by making phosphoenolpyruvate (PEP) available for perturbation studies with Escherichia coli producing L-phenylalanine. A production strain overexpressing a PEP-transporter variant (UhpT-D388C) is applied in a standardized fed-batch production-process on a 42 L-scale. Four parallel short-term perturbation experiments of 20 min are performed with glucose and glycerol as fed-batch carbon sources after rapid media transition of cells from the production-process. PEP is added after 9 min and is immediately consumed by the cells with up to 1.5 mmol gCDW-1 h-1 . L-phenylalanine production rates increased by up to 200% after addition of PEP. This clearly indicates an intracellular PEP-limitation in the L-phenylalanine production strain under study. Thus, it is shown that overexpressing specific transporters for analytical reasons makes exogenous substrates available as perturbation substrates for metabolic analyses of cells sampled from production-processes and thereby allows a very targeted perturbation of whole-cell metabolism.

Enhancement of l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin.

l-Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l-phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (aroF) and feedback-resistant chorismate mutase/prephenate dehydratase (pheAfbr ), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake-flask studies showed that vgb-expressing strains displayed higher rates of oxygen uptake, and l-phenylalanine production under standard aeration conditions was increased. In the aerobicfermentation process, cell growth, l-phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheAfbr , and tac-vgb genes, were increased compared to that in the strain harboring only aroF and pheAfbr (E. coli strain PAP), especially under oxygen-limited conditions. The vgb-expressing strain PAPV produced 21.9% more biomass and 16.6% more l-phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l-phenylalanine production by E. coli using less intensive and thus more economical aeration conditions.

Metabolic control analysis of l-phenylalanine production from glycerol with engineered E. coli using data from short-term steady-state perturbation experiments

The metabolic control of l-phenylalanine producing Escherichia coli cells was studied in a fed-batch process with glycerol as carbon source. To this end, metabolic perturbation experiments were performed in parallel to the operating process with cells harvested at two process times of interest: at the beginning of the l-phenylalanine production phase and at the end of the process with decreasing l-phenylalanine production. Data from the parallelised short-term perturbation experiments (19min) was used for the evaluation of microbial metabolome, fluxome and thermokinetics. This data was to determine local elasticity coefficients and subsequently the global flux control coefficients. Through the application of this methodical setup, several metabolic reactions were identified which control carbon flux towards l-phenylalanine: reactions that consume and produce phosphoenolpyruvate, while enzymes catalysing reactions of the glycerol metabolism were identified as being highly relevant for l-phenylalanine production. A significant change in metabolic control was detected by comparing the flux control coefficients during the two process phases. At decreasing l-phenylalanine production metabolic control appeared to be redirected towards a limiting fructose-6-phosphate and phosphoenolpyruvate supply. In all, due to the combination of an experimental approach and the framework of metabolic control analysis, improved knowledge about the metabolic network was generated within one single experiment.