L-Phe Production Research Articles

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.

Production of Natural 2-Phenylethanol from Glucose or Glycerol with CoupledEscherichia coliStrains Expressingl-Phenylalanine Biosynthesis Pathway and Artificial Biocascades

2-Phenylethanol (2-PE) is a fragrance widely used in food and cosmetics. Natural 2-PE is preferred in these applications but with high price and very limited availability. Microbial synthesis from glucose or glycerol is an attractive way to produce natural 2-PE, but suffers from low product titer. Escherichia coli NST74-Phe-Sty was engineered to coexpress the l-phenylalanine (l-Phe) biosynthesis pathway and enzyme cascade of l-Phe to 2-PE, producing only 0–0.17 g/L 2-PE from glucose at 22–37 °C due to the incompatibility of the temperature for enzyme expression and activity. Enhanced production of 2-PE (8.4–9.1 g/L) from glucose or glycerol was achieved by coupling of E. coli NST74-Phe expressing the l-Phe biosynthesis pathway for l-Phe production at the optimal temperature of 37 °C with E. coli T7-Sty expressing enzyme cascades of l-Phe to 2-PE at the optimal expression temperature of 22 °C and the optimal biotransformation temperature of 30 °C. The 2-PE titer is 4.7-fold higher than the best reported 2-PE concentration produced from glucose. The coupled strains approach could be generally applicable for enhancing microbial production of useful chemicals from sugars or glycerol.

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.

Biosensor-Based Evolution and Elucidation of a Biosynthetic Pathway in Escherichia coli.

The successful evolution of metabolite-producing microbes requires a high-throughput screening method to obtain the desired properties within a short time. In this study, we developed a transcription-factor-driven device that combines a metabolite-responsive element and a selection module. This device was able to specifically sense intracellular l-phenylalanine (l-Phe) and convert this signal into an observable phenotype. Applying this device, we successfully improved l-Phe production by screening hyperproducing phenotypes from a ribonucleotide binding site library and a random mutagenesis library. In addition, several site mutations introduced by random mutagenesis were identified and elucidated to facilitate the improvement of l-Phe production. Our results present a paradigm for screening of compounds that are not easily observable to raise the yield of targeted compounds from a large candidate library. This approach may guide further applications in rewiring metabolic circuits and facilitate the directed evolution of recombinant strains.

Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum.

Microbial production of L-phenylalanine (L-Phe) from renewable sources has attracted much attention recently. In the present study, Corynebacterium glutamicum 13032 was rationally engineered to produce L-Phe from inexpensive glucose. First, all the L-Phe biosynthesis pathway genes were investigated and the results demonstrated that in addition to AroF and PheA, the native PpsA, TktA, AroE and AroA, and the heterologous AroL and TyrB were also the key enzymes for L-Phe biosynthesis. Through combinational expression of these key enzymes, the L-Phe production was increased to 6.33 ± 0.13 g l(-1) which was about 1.48-fold of that of the parent strain C. glutamicum (pXM-pheA (fbr)-aroF (fbr)) (fbr, feedback-inhibition resistance). Furthermore, the production of L-Phe was improved to 9.14 ± 0.21 g l(-1) by modifying the glucose and L-Phe transport systems and blocking the acetate and lactate biosynthesis pathways. Eventually, the titer of L-Phe was enhanced to 15.76 ± 0.23 g l(-1) with a fed-batch fermentation strategy. To the best of our knowledge, this was the highest value reported in rationally engineered C. glutamicum 13032 strains. The results obtained will also contribute to rational engineering of C. glutamicum for production of other valuable aromatic compounds.

Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: the role of extra copies of glpK, glpX, and tktA genes.

BackgroundFor the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E. coli, is taken up via the PEP-dependent phosphotransferase system which delivers glucose-6-phosphate (G6P). G6P enters either glycolysis or the PPP. In contrast, glycerol is phosphorylated by an ATP-dependent glycerol kinase (GlpK) thus saving one PEP. However, two gluconeogenic reactions (fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, FBPase) are necessary for growth and provision of E4P. Glycerol has become an important carbon source for biotechnology and reports on production of L-Phe from glycerol are available. However, the influence of FBPase and transketolase reactions on L-Phe production has not been reported.ResultsL-Phe productivity of parent strain FUS4/pF81 (plasmid-encoded genes for aroF, aroB, aroL, pheA) was compared on glucose and glycerol as C sources. On glucose, a maximal carbon recovery of 0.19 mM CPhe/CGlucose and a maximal space-time-yield (STY) of 0.13 g l−1 h−1 was found. With glycerol, the maximal carbon recovery was nearly the same (0.18 mM CPhe/CGlycerol), but the maximal STY was higher (0.21 g l−1 h−1). We raised the chromosomal gene copy number of the genes glpK (encoding glycerol kinase), tktA (encoding transketolase), and glpX (encoding fructose-1,6-bisphosphatase) individually. Overexpression of glpK (or its feedback-resistant variant, glpKG232D) had little effect on growth rate; L-Phe production was about 30% lower than in FUS4/pF81. Whereas the overexpression of either glpX or tktA had minor effects on productivity (0.20 mM CPhe/CGlycerol; 0.25 g l−1 h−1 and 0.21 mM CPhe/CGlycerol; 0.23 g l−1 h−1, respectively), the combination of extra genes of glpX and tktA together led to an increase in maximal STY of about 80% (0.37 g l−1 h−1) and a carbon recovery of 0.26 mM CPhe/CGlycerol.ConclusionsEnhancing the gene copy numbers for glpX and tktA increased L-Phe productivity from glycerol without affecting growth rate. Engineering of glycerol metabolism towards L-Phe production in E. coli has to balance the pathways of gluconeogenesis, glycolysis, and PPP to improve the supply of the precursors, PEP and E4P.

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving L-phenylalanine (L-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of L-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of L-Phe. In contrast, overexpression of aroF (wt) or pheA (fbr) from E. coli significantly increased L-Phe production. Co-overexpression of aroF (wt) and pheA (fbr) improved the titer of L-Phe to 4.46 ± 0.06 g l⁻¹. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of L-Phe production. Co-overexpression of the mutated pheA (fbr) and the wild-type gene aroH resulted in the production of L-Phe up to 4.64 ± 0.09 g l⁻¹. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.

Design of a recombinant Escherichia coli for producing l-phenylalanine from glycerol

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L-phenylalanine (L-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of L-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L-Phe was only 76 % of the maximum value attained in the fermenter.

Experiment of L-Phenylalanine Fermentation in New Bioreactor

L-phenylalanine fermentation (L-Phe) is produced by the preparation method of PPA-transamination using the ferment liquor, which is the production of the E.coli No.1 fermented in heat pipe bioreactor (HPBR), the technical indexes are superior. Compared with in the traditional agitated bioreactor, the biomass(taken its measure by OD value) of microbe fermented in new bioreactor is enhanced from 0.40 to 0.54, moreover, the average rate of L-Phe production by the preparation method of PPA-transamination is raised from 70% to 75%.

Comparative study of L-phenylalanine production in the growing and stationary phases during high cell density cultivation of an auxotrophic Escherichia coli

High cell density cultivation was investigated for L-phenylalanine (L-Phe) production by an L-tyrosine (L-Tyr) auxotrophic Escherichia coli WSH-BR165 (pAPB03). Dual exponential feeding of L-Tyr and glucose was adopted to achieve high cell density cultivation. The control was completed without dual feeding. The processes where dual feeding and induction were commenced together and those where induction began after dual feeding were studied and compared. The results indicated that the former dual feeding mode was most favorable for enhanced L-Phe production. With an optimal specific growth rate of 0.09/h during the dual exponential feeding period, the maximum dry cell weight reached 43.16 g/L (3.04 times that of the control) with a final L-Phe titer of 44.53 g/L (1.06 times that of the control) and an L-Phe productivity of 1.484 g/L/h (1.69 times that of the control). High cell density cultivation via the feeding of L-Tyr and glucose exponentially after the induction point proved to be an efficient approach to enhance L-Phe production.

Enhanced l-phenylalanine production by recombinant Escherichia coli BR-42 (pAP-B03) resistant to bacteriophage BP-1 via a two-stage feeding approach

The L-phenylalanine (L-Phe) production by Escherichia coli WSH-Z06 (pAP-B03) was frequently prevented by bacteriophage BP-1 infestation. To cope with the bacteriophage BP-1 problem for an improved L-Phe production, one bacteriophage BP-1-resistant mutant, E. coli BR-42, was obtained from 416 mutant colonies of E. coli WSH-Z06 after N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis by selection for resistance to bacteriophage BP-1. The recombinant E. coli BR-42-carrying plasmid pAP-B03 had a high capacity in L-Phe production and a remarkable tolerance to 1 × 10(10) pfu (plaque-forming unit)/ml bacteriophage stock. For an enhanced L-Phe production by E. coli BR-42 (pAP-B03), the effects of different feeding strategies including pH-stat, constant rate feeding, linear decreasing rate feeding, and exponential feeding on L-Phe production were investigated; and a two-stage feeding strategy, namely exponential feeding at μ (set) = 0.18 h(-1) in the first 20 h and a following linear varying rate feeding with F = (-0.55 × t + 18.6) ml/h, was developed to improve L-Phe production. With this two-stage feeding approach, a maximum L-Phe titer of 57.63 g/l with a high L-Phe productivity (1.15 g/l/h) was achieved, which was 15% higher than the highest level (50 g/l) reported so far according to our knowledge. The recombinant E. coli BR-42 (pAP-B03) is a potential L-Phe over-producer in substantial prevention of bacteriophage BP-1 infestation compared to its parent strain WSH-Z06 (pAP-B03).

Modeling and Optimization of Phenylalanine Ammonia Lyase Stabilization in Recombinant Escherichia coli for the Continuous Synthesis of l-Phenylalanine on the Statistical-Based Experimental Designs

Some approaches for improving recombinant phenylalanine ammonia lyase (PAL) stability in Escherichia coli during the enzymatic methods of l-phenylalanine (l-Phe) production were developed following preliminary studies by means of statistical-based experiment designs (response surface method). The traditional non-statistical technology was used to screen four critical factors for PAL stability during the bioconversion process, viz., glycerin, sucrose, 1,4-dithiothreitol (DTT), and MgSO(4). The central composite design (CCD) was applied to optimize the combined effect of critical factors for recombinant PAL stability and understand the relationship between the factors and PAL stability. The optimum values for testing variables were 13.04 mM glycerin, 1.87 mM sucrose, 4.09 mM DTT, and 69 mM Mg(2+). A second-order model equation was suggested and then validated experimentally. The model adequacy was very satisfactory because the coefficient of determination was 0.88. The maximum PAL activity was retained as 67.73 units/g after three successive cycles of bioconversion. In comparison to initial PAL activity, the loss of PAL activity was only 22%. PAL activity was enhanced about 23% in comparison to the control (without any stabilizer additives). PAL stability was significantly improved during successive bioconversion. The results obtained here verified the effectiveness of the applied methodology and may be helpful for l-Phe production on an industrial scale.

Metabolic transcription analysis of engineered Escherichia coli strains that overproduce L-phenylalanine

BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale.

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.

Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12.

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.

Production of L-phenylalanine from acetamidocinnamic acid usingBacillus sphaericus andParacoccus denitrificans

To establish an efficient production method for L-phenylalanine (L-Phe), the possibility of L-Phe production from acetamidocinnamic acid by a two-step enzyme reaction was investigated.Bacillus sphaericus N-7 (FERM-P no. 6746) was selected as an enzyme source for the acylase used to form phenylpyruvic acid from aceta midocinnamic acid.Paracoccus denitrificans IFO 12442 was selected as an aminotransferase source for the conversion of phenylpyruvic acid to L-Phe. By using both cell types, 0.46M L-Phe was produced in a molar yield of 92% from 0.5M acetamidocinnamic acid.