ABSTRACT
Phenylalanine (Phe) is the precursor of essential secondary products in plants. Here we show that a key, rate-limiting step in Phe biosynthesis, which is catalyzed by arogenate dehydratase, experienced feedback de-regulation during evolution. Enzymes from microorganisms and type-I ADTs from plants are strongly feedback-inhibited by Phe, while type-II isoforms remain active at high levels of Phe. We have found that type-II ADTs are widespread across seed plants and their overproduction resulted in a dramatic accumulation of Phe in planta, reaching levels up to 40 times higher than those observed following the expression of type-I enzymes. Punctual changes in the allosteric binding site of Phe and adjacent region are responsible for the observed relaxed regulation. The phylogeny of plant ADTs evidences that the emergence of type-II isoforms with relaxed regulation occurred at some point in the transition between nonvascular plants and tracheophytes, enabling the massive production of Phe-derived compounds, primarily lignin, a hallmark of vascular plants.
Subject(s)
Crops, Agricultural/genetics , Evolution, Molecular , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Phenylalanine/biosynthesis , Phenylalanine/genetics , Plants/genetics , Arabidopsis/genetics , Arabidopsis/metabolism , Crops, Agricultural/metabolism , Cucumis sativus/genetics , Cucumis sativus/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Oryza/genetics , Oryza/metabolism , Phaseolus/genetics , Phaseolus/metabolism , Phylogeny , Nicotiana/genetics , Nicotiana/metabolism , Zea mays/genetics , Zea mays/metabolismABSTRACT
Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.
Subject(s)
Hydro-Lyases/genetics , Petunia/genetics , Petunia/metabolism , Phenylalanine/biosynthesis , Plant Proteins/genetics , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Flowers/genetics , Flowers/metabolism , Gene Expression Regulation, Plant , Hydro-Lyases/metabolism , Mutation , Phenylalanine/metabolism , Plant Proteins/metabolism , Plants, Genetically Modified , Plastids/genetics , Plastids/metabolism , Secondary Metabolism/genetics , Shikimic Acid/metabolismABSTRACT
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.
Subject(s)
Coumaric Acids/chemical synthesis , Coumaric Acids/economics , Coumaric Acids/isolation & purification , Benzaldehydes/chemistry , Biomass , Escherichia coli/chemistry , Escherichia coli/genetics , Microwaves , Molecular Structure , Phenylalanine/biosynthesis , Phenylalanine/chemistry , Plant Extracts/chemistry , Plants/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Tyrosine/biosynthesis , Tyrosine/chemistryABSTRACT
In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.
Subject(s)
Indoleacetic Acids/chemistry , Indoleacetic Acids/metabolism , Phenylalanine/biosynthesis , Biosynthetic Pathways/genetics , Cytosol/metabolism , Indoles , Phenylalanine/metabolism , Phenylpyruvic Acids/metabolism , Plants/metabolism , TryptophanABSTRACT
Escherichia coli, a model microorganism for which convenient metabolic engineering tools are available and that grows quickly in cheap media, has been widely used in the production of valuable chemicals, including aromatic amino acids. As the three aromatic amino acids, L-tryptophan, L-tyrosine, and L-phenylalanine, share the same precursors, to increase the titer of a specific aromatic amino acid, the branch pathways to the others are usually permanently inactivated, which leads to the generation of auxotrophic strains. In this study, a tunable switch that can toggle between different states was constructed. Then, a switchable and non-auxotrophic E. coli strain for synthesis of aromatic amino acids was constructed using this tunable switch. By adding different inducers to cultures, three different production patterns of aromatic amino acids by the engineered strain could be observed. This tunable switch can also be applied in regulating other branch pathways and in other bacteria.
Subject(s)
Escherichia coli/metabolism , Phenylalanine/biosynthesis , Tryptophan/biosynthesis , Tyrosine/biosynthesis , Escherichia coli/genetics , Metabolic EngineeringABSTRACT
In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904⯱â¯53 mg/gDW (580⯱â¯34â¯mg/L) of phenylalanine and 64⯱â¯3.7 mg/gDW (41⯱â¯2.3â¯mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470⯱â¯70 mg/gDW (207â¯mg/L) of p-coumaric acid, 267⯱â¯31 mg/gDW (114â¯mg/L) of cinnamic acid and 47.4⯱â¯13.9 mg/gDW (12.6â¯mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.
Subject(s)
Metabolic Engineering , Phenylalanine , Phenylpropionates/metabolism , Synechocystis , Tyrosine , Escherichia coli/genetics , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Phenylalanine/biosynthesis , Phenylalanine/genetics , Synechocystis/genetics , Synechocystis/growth & development , Tyrosine/biosynthesis , Tyrosine/geneticsABSTRACT
The biocatalytic synthesis of L- and D-phenylalanine analogues of high synthetic value have been developed using as biocatalysts mutant variants of phenylalanine ammonia lyase from Petroselinum crispum (PcPAL), specifically tailored towards mono-substituted phenylalanine and cinnamic acid substrates. The catalytic performance of the engineered PcPAL variants was optimized within the ammonia elimination and ammonia addition reactions, focusing on the effect of substrate concentration, biocatalyst:substrate ratio, reaction buffer and reaction time, on the conversion and enantiomeric excess values. The optimal conditions provided an efficient preparative scale biocatalytic procedure of valuable phenylalanines, such as (S)-m-methoxyphenylalanine (Y = 40%, ee > 99%), (S)-p-bromophenylalanine (Y = 82%, ee > 99%), (S)-m-(trifluoromethyl)phenylalanine (Y = 26%, ee > 99%), (R)-p-methylphenylalanine, (Y = 49%, ee = 95%) and (R)-m-(trifluoromethyl)phenylalanine (Y = 34%, ee = 93%).
Subject(s)
Petroselinum/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Phenylalanine/biosynthesis , Ammonia/metabolism , Biocatalysis , Biotransformation , Genetic Engineering , Petroselinum/enzymology , Petroselinum/genetics , Phenylalanine Ammonia-Lyase/geneticsABSTRACT
Plants produce numerous natural products that are essential to both plant and human physiology. Recent identification of genes and enzymes involved in their biosynthesis now provides exciting opportunities to reconstruct plant natural product pathways in heterologous systems through synthetic biology. The use of plant chassis, although still in infancy, can take advantage of plant cells' inherent capacity to synthesize and store various phytochemicals. Also, large-scale plant biomass production systems, driven by photosynthetic energy production and carbon fixation, could be harnessed for industrial-scale production of natural products. However, little is known about which plants could serve as ideal hosts and how to optimize plant primary metabolism to efficiently provide precursors for the synthesis of desirable downstream natural products or specialized (secondary) metabolites. Although primary metabolism is generally assumed to be conserved, unlike the highly-diversified specialized metabolism, primary metabolic pathways and enzymes can differ between microbes and plants and also among different plants, especially at the interface between primary and specialized metabolisms. This review highlights examples of the diversity in plant primary metabolism and discusses how we can utilize these variations in plant synthetic biology. I propose that understanding the evolutionary, biochemical, genetic, and molecular bases of primary metabolic diversity could provide rational strategies for identifying suitable plant hosts and for further optimizing primary metabolism for sizable production of natural and bio-based products in plants.
Subject(s)
Biological Evolution , Plants/metabolism , Amino Acids/biosynthesis , Biological Products/metabolism , Metabolic Engineering , Metabolic Networks and Pathways , Mevalonic Acid/metabolism , Phenylalanine/biosynthesis , Plant Proteins/metabolism , Plants/genetics , Substrate SpecificityABSTRACT
Communities of bacteria that cause Legionnaires' disease repel other bacteria by secreting an acid called HGA.
Subject(s)
Legionella pneumophila/metabolism , Legionnaires' Disease/metabolism , Phenylalanine/metabolism , Tyrosine/metabolism , Environmental Microbiology , Humans , Legionella pneumophila/genetics , Legionella pneumophila/pathogenicity , Legionnaires' Disease/microbiology , Phenylalanine/biosynthesis , Tyrosine/biosynthesisABSTRACT
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.
Subject(s)
Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Industrial Microbiology/methods , Metabolic Engineering/methods , Phenylalanine/biosynthesis , Amino Acid Transport Systems, Neutral/genetics , Biosynthetic Pathways/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Fermentation , Glucose/metabolism , Metabolic Flux Analysis , Mutation , Phenylalanine/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Promoter Regions, GeneticABSTRACT
This work aims to uncover how glucose affected the production of phenyllactic acid (PLA) and p-hydroxyphenyllactic acid ( p-OH-PLA). The highest yields of PLA (68.53 mg/L) and p-OH-PLA (50.39 mg/L) were observed after Lactobacillus plantarum strain YM-4-3 fermentation in media containing 30 and 10 g/L glucose, respectively. Additionally, the antimicrobial activity of YM-4-3 against food-borne pathogens and the NADH/NAD+ ratio were positively correlated with the production of PLA and p-OH-PLA, respectively. In addition, a 2-oxoglutarate/malate translocator coding gene ( Omt1) was selected based on the qPCR results, and its knockout mutant, compared with the wild-type strain YM-4-3, showed that the PLA and p-OH-PLA production was decreased by 1.37-6.99 and 1.53-1.59 times, respectively. This result indicated that OMT1 was involved in the biosynthesis of PLA and p-OH-PLA. To conclude, this study suggests that glucose, NADH/NAD+ ratio and/or the Omt1 gene, PLA, and p-OH-PLA production, and antimicrobial activity contribute to a cause-and-effect relationship.
Subject(s)
Anti-Bacterial Agents/metabolism , Antifungal Agents/metabolism , Bacterial Proteins/metabolism , Glucose/metabolism , Ketoglutaric Acids/metabolism , Lactates/metabolism , Lactobacillus plantarum/metabolism , Malates/metabolism , Membrane Transport Proteins/metabolism , Phenylalanine/analogs & derivatives , Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Bacteria/drug effects , Bacterial Proteins/genetics , Fermentation , Food Microbiology , Fungi/drug effects , Lactates/pharmacology , Lactobacillus plantarum/genetics , Membrane Transport Proteins/genetics , Phenylalanine/biosynthesisABSTRACT
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.
Subject(s)
Brain Ischemia/metabolism , Chalcone/analogs & derivatives , Mitochondria/drug effects , Mitochondria/metabolism , Neuroprotective Agents/pharmacology , Phenylalanine/biosynthesis , Quinones/pharmacology , Reperfusion Injury/metabolism , Animals , Behavior, Animal/drug effects , Brain Ischemia/drug therapy , Brain Ischemia/etiology , Brain Ischemia/pathology , Chalcone/pharmacology , Disease Models, Animal , Energy Metabolism/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Mice , Neurons/drug effects , Neurons/metabolism , Organelle Biogenesis , Oxidative Stress/drug effects , PC12 Cells , Rats , Reperfusion Injury/drug therapy , Reperfusion Injury/etiology , Reperfusion Injury/pathology , Treatment OutcomeABSTRACT
BACKGROUND: L-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. RESULTS: We 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. CONCLUSION: The 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.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Engineering/methods , Phenylalanine/biosynthesis , Bioreactors , Biotechnology/instrumentation , Biotechnology/methods , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Fermentation , Gene Expression Regulation, Bacterial , Glucose/metabolism , Metabolic Networks and Pathways/genetics , Microorganisms, Genetically-Modified , Mutation , Phenylalanine/genetics , Phosphoenolpyruvate/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Proteomics/methods , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
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 aerobic fermentation 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.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Phenylalanine/biosynthesis , Truncated Hemoglobins/genetics , Truncated Hemoglobins/metabolism , Bacterial Proteins/biosynthesis , Fermentation , Phenylalanine/chemistry , Phenylalanine/genetics , Promoter Regions, Genetic/genetics , Truncated Hemoglobins/biosynthesisABSTRACT
Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level. These characteristics impose several challenges for tailoring it, and various modifications need to be applied in order to redirect the carbon flux towards the production of the desired compounds. This minireview addresses the metabolic engineering approaches targeting the central carbon metabolism, the shikimate pathway and the tyrosine and phenylalanine biosynthetic pathway of S. cerevisiae for biosynthesis of aromatic chemicals and their derivatives from glucose.
Subject(s)
Glucose/metabolism , Hydrocarbons, Aromatic/metabolism , Metabolic Engineering/methods , Metabolic Networks and Pathways/genetics , Saccharomyces cerevisiae/metabolism , Biotransformation , Chorismic Acid/biosynthesis , Fermentation , Industrial Microbiology , Phenylalanine/biosynthesis , Saccharomyces cerevisiae/genetics , Shikimic Acid/metabolism , Tyrosine/biosynthesisABSTRACT
The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG fbr performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications. Knocking in the tktA gene under a tyrR promoter and knocking out pykF further increased phenylalanine production demonstrating that the supply of precursor via PEP and E4P is also a rate-limiting step. Finally, we tested the role of global regulators on this deregulated pathway and found that Fis overexpression helps in both enhancing and sustaining the flux through this pathway. This work opens up the possibility of using global regulators in synergy with pathway-specific modifications to enhance product yields.
Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Enhancement/methods , Metabolic Engineering/methods , Models, Biological , Phenylalanine/biosynthesis , Computer Simulation , Metabolic Flux Analysis/methods , Metabolic Networks and Pathways/physiology , Phenylalanine/isolation & purification , Up-Regulation/geneticsABSTRACT
BACKGROUND: As the fastest growing plant, duckweed can thrive on anthropogenic wastewater. The purple-backed duckweed, Landoltia punctata, is rich in starch and flavonoids. However, the molecular biological basis of high flavonoid and low lignin content remains largely unknown, as does the best method to combine nutrients removed from sewage and the utilization value improvement of duckweed biomass. RESULTS: A combined omics study was performed to investigate the biosynthesis of flavonoid and the metabolic flux changes in L. punctata grown in different culture medium. Phenylalanine metabolism related transcripts were identified and carefully analyzed. Expression quantification results showed that most of the flavonoid biosynthetic transcripts were relatively highly expressed, while most lignin-related transcripts were poorly expressed or failed to be detected by iTRAQ based proteomic analyses. This explains why duckweed has a much lower lignin percentage and higher flavonoid content than most other plants. Growing in distilled water, expression of most flavonoid-related transcripts were increased, while most were decreased in uniconazole treated L. punctata (1/6 × Hoagland + 800 mgâ¢L-1 uniconazole). When L. punctata was cultivated in full nutrient medium (1/6 × Hoagland), more than half of these transcripts were increased, however others were suppressed. Metabolome results showed that a total of 20 flavonoid compounds were separated by HPLC in L. punctata grown in uniconazole and full nutrient medium. The quantities of all 20 compounds were decreased by uniconazole, while 11 were increased and 6 decreased when grown in full nutrient medium. Nutrient starvation resulted in an obvious purple accumulation on the underside of each frond. CONCLUSIONS: The high flavonoid and low lignin content of L. punctata appears to be predominantly caused by the flavonoid-directed metabolic flux. Nutrient starvation is the best option to obtain high starch and flavonoid accumulation simultaneously in a short time for biofuels fermentation and natural products isolation.
Subject(s)
Araceae/metabolism , Flavonoids/metabolism , Starch/metabolism , Araceae/genetics , Gene Expression Regulation, Plant , Lignin/biosynthesis , Phenylalanine/biosynthesis , Proteomics , Sequence Analysis, RNAABSTRACT
L-Phenylalanine (L-Phe) is an important amino acid used in both food and medicinal applications. We developed an in vitro system that allowed a direct, quantitative investigation of phenylalanine biosynthesis in E. coli. Here, the absolute concentrations of six enzymes (AroK, AroL, AroA, AroC, PheA and TyrB) involved in the shikimate (SHIK) pathway were determined by a quantitative proteomics approach and in vitro enzyme titration experiments. The reconstitution of an in vitro reaction system for these six enzymes was established and their effects on the phenylalanine production were tested. The results showed that the yield of phenylalanine increased 3.0 and 2.1 times when the concentrations of shikimate kinase (AroL) and 5-enolpyruvoyl shikimate 3-phosphate (EPSP) synthase (AroA) were increased 2.5 times. Consistent results were obtained from in vivo via the overexpression of AroA in a phenylalanine-producing strain, and the titer of phenylalanine reached 62.47 g/l after 48 h cultivation in a 5-liter jar fermentor. Our quantitative findings provide a practical method to detect the potential bottleneck in a specific metabolic pathway to determine which gene products should be targeted to improve the yield of the desired product.
Subject(s)
Enzymes/metabolism , Escherichia coli/metabolism , Phenylalanine/biosynthesis , Proteomics/methods , 3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism , Enzymes/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Phenylalanine/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Reproducibility of Results , Shikimic Acid/metabolismABSTRACT
Aromatic compounds such as l-phenylalanine, 2-phenylethanol and trans-cinnamate are aromatic compounds of industrial interest. Current trends support replacement of chemical synthesis of these compounds by 'green' alternatives produced in microbial cell factories. The solvent-tolerant Pseudomonas putida DOT-T1E strain was genetically modified to produce up to 1 g l-1 of l-phenylalanine. In order to engineer this strain, we carried out the following stepwise process: (1) we selected random mutants that are resistant to toxic phenylalanine analogues; (2) we then deleted up to five genes belonging to phenylalanine metabolism pathways, which greatly diminished the internal metabolism of phenylalanine; and (3) in these mutants, we overexpressed the pheAfbr gene, which encodes a recombinant variant of PheA that is insensitive to feedback inhibition by phenylalanine. Furthermore, by introducing new genes, we were able to further extend the diversity of compounds produced. Introduction of histidinol phosphate transferase (PP_0967), phenylpyruvate decarboxylase (kdc) and an alcohol dehydrogenase (adh) enabled the strain to produce up to 180 mg l-1 2-phenylethanol. When phenylalanine ammonia lyase (pal) was introduced, the resulting strain produced up to 200 mg l-1 of trans-cinnamate. These results demonstrate that P. putida can serve as a promising microbial cell factory for the production of l-phenylalanine and related compounds.
Subject(s)
Cinnamates/metabolism , Flavoring Agents/metabolism , Phenylalanine/biosynthesis , Phenylethyl Alcohol/metabolism , Pseudomonas putida/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Industrial Microbiology , Pseudomonas putida/geneticsABSTRACT
Phenylalanine dehydrogenase (PheDH) plays an important role in enzymatic synthesis of L-phenylalanine for aspartame (sweetener) and detection of phenylketonuria (PKU), suggesting that it is important to obtain a PheDH with excellent characteristics. Gene fusion of PheDH and formate dehydrogenase (FDH) was constructed to form bifunctional multi-enzymes for bioconversion of L-phenylalanine coupled with coenzyme regeneration. Comparing with the PheDH monomer from Microbacterium sp., the bifunctional PheDH-FDH showed noteworthy stability under weakly acidic and alkaline conditions (pH 6.5-9.0). The bifunctional enzyme can produce 153.9 mM L-phenylalanine with remarkable performance of enantiomers choice by enzymatic conversion with high molecular conversion rate (99.87 %) in catalyzing phenylpyruvic acid to L-phenylalanine being 1.50-fold higher than that of the separate expression system. The results indicated the potential application of the PheDH and PheDH-FDH with coenzyme regeneration for phenylpyruvic acid analysis and L-phenylalanine biosynthesis in medical diagnosis and pharmaceutical field.