Production Of L-tryptophan Research Articles

Optimization of L-Tryptophan Biosynthesis From L-Serine of Processed Iranian Beet and Cane Molasses and Indole by Induced Escherichia coli ATCC 11303 Cells.

Background:L-tryptophan is an important ingredient in medicines, especially in neuromedicines such as antidepressants. Many commercial processes employ various microorganisms with high tryptophan synthase activity to produce L-tryptophan from indole and L-serine, but these processes are very costly due to the costs of precursors, especially L-serine.Objectives:For this reason, we studied the ability to use processed Iranian cane and beet molasses as L-serine sources for L-tryptophan production, which enables us to reach a cost-effective process.Materials and Methods:Whole cells of Escherichia coli ATCC 11303 were induced for L-tryptophan synthase by addition of indole to the growth medium and bacterial cells harvested from the growth medium were used as biocatalysts in the production medium. Conditions of the production medium were optimized and Iranian cane and beet molasses were processed by solvent extraction with ethanol and n-butanol and used as L-serine sources of the production medium. Amount of L-tryptophan and theoretical yield of L-tryptophan production were determined by High Performance Liquid Chromatography and by a colorimetrical method on the basis of the remaining indole assay, respectively.Results:L-tryptophan production increased by 15 folds, when indole was used as an inducer. L-tryptophan was produced from processed Iranian beet molasses in satisfactory amounts (0.53 mM) and no exogenous pyridoxal phosphate was required as a cofactor under our experimental conditions.Conclusions:The obtained results proved that Iranian beet molasses include significant amounts of L-serine that makes them a suitable substitution for L-serine. Findings of the present study give impetus to use of Iranian beet molasses for cost-effective L-Trp production in the amino acid industry.

Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on L-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing L-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular L-tryptophan. In the L-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of L-tryptophan (48.68g/L) was obtained, which represented a 15.96% increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95g/L. The intracellular concentration of L-tryptophan was low, and the glucose conversion rate reached a high level of 21.87%, which was increased by 15.53% as compared with the parent strain.

Knocking out analysis of tryptophan permeases in Escherichia coli for improving L-tryptophan production

Three permeases, Mtr, TnaB, and AroP, are involved in the uptake of L-tryptophan in Escherichia coli. These permeases possess individual function for cell transportation and metabolism, and affect extracellular L-tryptophan accumulation. In this study, by knocking out three tryptophan permeases separately and simultaneously in L-tryptophan-producing strain E. coli GPT1002, we analyzed the effect of permease knock out on L-tryptophan uptake, cell growth, and L-tryptophan production. We found that TnaB is the main transporter that is responsible for the uptake of L-tryptophan. Inactivation of tnaB improved the L-tryptophan production significantly, and inactivation of aroP has an additive effect on tnaB mutant. Quantitative real-time PCR analysis confirmed that knocking out permeases affects gene transcription and cell metabolism in many metabolic pathways. The tryptophan permease-deficient GPT1017 mutant exhibited the highest L-tryptophan production at 2.79 g l(-1), which is 51.6 % higher than that produced by the control strain. In 5-l bioreactor fermentation, the L-tryptophan production in GPT1017 reached 16.3 g l(-1).

Catalytic resolution of dl-tryptophan amides using the resting cells of Flavobacterium aquatile ZJB-09211 in a two-phase system

The catalytic activity of Flavobacterium aquatile ZJB-09211 towards the kinetic resolution of DL-tryptophan amides was significantly enhanced by ethyl acetate. A maximum enzyme activity of 5118.62 U/g was obtained under the optimized conditions consisting of a mixture of ethyl acetate and Tris–HCl buffer (30:70). In a scale-up reaction, the tryptophan amide concentration was improved to 200 mM, with 49.85% ( e.e. > 99.95%) of the substrate being converted to l -tryptophan. The addition of an organic solvent to the process therefore provided an effective approach for improving the activity of the amidase that could be applied to other amidase-catalyzed bioprocesses. • Ethyl acetate enhanced the activity of amidase and facilitated product separation. • Space-time yield of the bioprocess was improved in this two-phase reaction system. • This methodology may play a pivot role in large scale production of L-tryptophan.

Enhanced production of l-tryptophan with glucose feeding and surfactant addition and related metabolic flux redistribution in the recombinant Escherichia coli

The production of l-tryptophan was investigated in a recombinant strain Escherichia coli W3110-ZDrr. It was observed that phosphate and feeding strategies are key factors to ensure the good cell growth and high production of l-tryptophan. The simple exponential feeding strategy could only produce 10.6 g/L l-tryptophan due to the improper feeding rate; while the manual glucose-feedback feeding approach could effectively control the substrate and inhibit the formation of acetate, and thus improvemed the l-tryptophan production to 25.5 g/L. The modified exponential feeding approach avoided overfeeding or underfeeding and achieved high production of l-tryptophan. Moreover, the addition of Tween 60 or PL61 could enhance the cell growth and the production of l-tryptophan in the fed-batch mode. Subsequent metabolic flux analysis showed that more carbon flux was distributed into the biosynthesis of l-tryptophan when Tween 60 or PL61 was supplied. The present work presents one base for further large-scale production of this important amino acid.

Modification of histidine biosynthesis pathway genes and the impact on production of l-histidine in Corynebacterium glutamicum

Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18hisDPtac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The L-histidine titer at 0.85 g l(-1) was 80 % greater in the transformed bacterium and production of byproducts, L-alanine and L-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l(-1)). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production.

The improved l-tryptophan production in recombinant Escherichia coli by expressing the polyhydroxybutyrate synthesis pathway

Polyhydroxybutyrate (PHB), the best known polyhydroxyalkanoates (PHA) has been believed to change intracellular metabolic flow and oxidation/reduction state, as well as enhance stress resistance of the host. In this study, a PHB biosynthesis pathway, which contains phaCAB operon genes from Ralstonia eutropha, was introduced into an L-tryptophan producing Escherichia coli strain GPT1002. The expression of the PHB biosynthesis genes resulted in PHB accumulation inside the cells and improved the L-tryptophan production. Quantitative real-time PCR analysis showed that the transcription of tryptophan operon genes in GPT2000 increased by 1.9 to 4.3 times compared with the control, indicating that PHB biosynthesis in engineered E. coli changed the physiological state of the host. Xylose was added into the medium as co-substrate to enhance the precursor supply for PHB biosynthesis. The addition of xylose improved both extracellular L-tryptophan production and intracellular PHB accumulation. Moreover, we obtained 14.4 g l(-1) L-tryptophan production and 9.7 % PHB (w/w) accumulation in GPT2000 via fed-batch cultivation.

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase

Acetate is a primary inhibitory metabolite in Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be derived from acetyl coenzyme A by the phosphotransacetylase (Pta)–acetate kinase (AckA) pathway. In this study, the fermentation characteristics of Pta mutant strain E. coli TRTHΔpta were compared with those of the control strain E. coli TRTH in a 30-L fermentor. The effects of glucose concentration and dissolved oxygen (DO) level were investigated, and the results suggest that DO and glucose concentration are vital influencing parameters for the production of L-tryptophan. Based on our experimental results, we then tested a DO-stat fed-batch fermentation strategy. When DO was controlled at about 20 % during L-tryptophan fermentation in the DO-stat fed-batch system, the pta mutant was able to maintain a higher growth rate at the exponential phase, and the final biomass and L-tryptophan production were increased to 55.3 g/L and 35.2 g/L, respectively. Concomitantly, as the concentration of acetate decreased to 0.7 g/L, the accumulation of pyruvate and lactate increased in the mutant strain as compared with the control strain. This characterization of the recombinant mutant strain provides useful information for the rational modification of metabolic fluxes to improve tryptophan production.

Strategy for pH control and pH feedback-controlled substrate feeding for high-level production of l-tryptophan by Escherichia coli

Optimum production of L-tryptophan by Escherichia coli depends on pH. Here, we established conditions for optimizing the production of L-tryptophan. The optimum pH range was 6.5-7.2, and pH was controlled using a three-stage strategy [pH 6.5 (0-12 h), pH 6.8 (12-24 h), and pH 7.2 (24-38 h)]. Specifically, ammonium hydroxide was used to adjust pH during the initial 24 h, and potassium hydroxide and ammonium hydroxide (1:2, v/v) were used to adjust pH during 24-38 h. Under these conditions, NH4 (+) and K(+) concentrations were kept below the threshold for inhibiting L-tryptophan production. Optimization was also accomplished using ratios (v/v) of glucose to alkali solutions equal to 4:1 (5-24 h) and 6:1 (24-38 h). The concentration of glucose and the pH were controlled by adjusting the pH automatically. Applying a pH-feedback feeding method, the steady-state concentration of glucose was maintained at approximately 0.2 ± 0.02 g/l, and acetic acid accumulated to a concentration of 1.15 ± 0.03 g/l, and the plasmid stability was 98 ± 0.5 %. The final, optimized concentration of L-tryptophan was 43.65 ± 0.29 g/l from 52.43 ± 0.38 g/l dry cell weight.

Production of l-tryptophan by enantioselective hydrolysis of d,l-tryptophanamide using a newly isolated bacterium

AbstractBacterial strain ZJB-09211 capable of amidase production has recently been isolated from soil samples. The strain is able to asymmetrically hydrolyze l-tryptophanamide from d,l-tryptophanamide to produce l-tryptophan in high yield and with excellent stereoselectivity (enantiomeric excess > 99.9 %, and enantiomeric ratio > 200). Strain ZJB-09211 has been identified as Flavobacterium aquatile based on the cell morphology analysis, physiological tests, and the 16S rDNA sequence analysis. Optimization of the fermentation medium led to an about six-fold increase in the amidase activity of strain ZJB-09211, which reached 501.5 U L−1. Substrate specifity and stereoselectivity investigations revealed that amidase of F. aquatile possessed a broad substrate spectrum and high enantioselectivity.

Effects of aroP gene disruption on L-tryptophan fermentation

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in Ltryptophan producing strains has not been fully explored. In this study, the aroP gene of the L-tryptophan producing Escherichia coli TRTH strain was disrupted using Red recombination technology and an aroP mutant E. coli TRTH ΔaroP was constructed. Fed-batch fermentation of E. coli TRTH ΔaroP was carried out in 30-L fermentor to investigate the L-tryptophan production. Compared with E. coli TRTH, the aroP mutant was able to maintain a higher growth rate during the exponential phase of the fermentation and the L-tryptophan production increased by 13.3%.

Modification of tryptophan transport system and its impact on production of l-tryptophan in Escherichia coli

The production of l-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of transport system for aromatic amino acids in l-tryptophan producing strains has not been fully explored. In this study, the fact was revealed that l-tryptophan production and cell growth were affected by the modification of transport systems based on YddG functioning as aromatic amino acid excretion and AroP functioning as general aromatic amino acid permease. Through comparing glucose conversion rates of recombinant strains such as Escherichia coli TRTH ΔaroP, E. coli TRTH-Y, and E. coli TRTH ΔaroP-Y, the moderate modification of transport system resulted in the metabolic flux redistribution of l-tryptophan biosynthesis pathway. In the fed-batch fermentation by E. coli TRTH and E. coli TRTH-Y in 30-liter fermentor, the final production of l-tryptophan fermented by E. coli TRTH-Y was 36.3g/L, which was 12.6% higher than fermentation by E. coli TRTH.

One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli

BackgroundL-tryptophan is an aromatic amino acid widely used in the food, chemical and pharmaceutical industries. In Escherichia coli, L-tryptophan is synthesized from phosphoenolpyruvate and erythrose 4-phosphate by enzymes in the shikimate pathway and L-tryptophan branch pathway, while L-serine and phosphoribosylpyrophosphate are also involved in L-tryptophan synthesis. In order to construct a microbial strain for efficient L-tryptophan production from glucose, we developed a one step tryptophan attenuator inactivation and promoter swapping strategy for metabolic flux optimization after a base strain was obtained by overexpressing the tktA, mutated trpE and aroG genes and inactivating a series of competitive steps.ResultsThe engineered E. coli GPT1002 with tryptophan attenuator inactivation and tryptophan operon promoter substitution exhibited 1.67 ~ 9.29 times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated 1.70 g l-1 L-tryptophan after 36 h batch cultivation in 300-mL shake flask. Bioreactor fermentation experiments showed that GPT1002 could produce 10.15 g l-1 L-tryptophan in 48 h.ConclusionsThe one step inactivating and promoter swapping is an efficient method for metabolic engineering. This method can also be applied in other bacteria.

Effect of feeding strategy on l-tryptophan production by recombinant Escherichia coli

Acetic acid is a primary inhibitory metabolite in Escherichia coli cultivation, and is detrimental to bacterial growth and formation of desired products. The effects of acetic acid on l-tryptophan production by recombinant E. coli were evaluated in this study. Results indicated that growth of an l-tryptophan-producing bacterium and l-tryptophan formation were inhibited significantly by acetic acid concentrations above 2 g/L. Different feeding strategies, including exponential feeding, pseudo-exponential feeding, and feeding based on DO-stat and glucose-stat were conducted and evaluated for l-tryptophan fermentation in 30-L jar fermenters. It was found that the maximum specific growth rate should be controlled below 0.25 h−1 in a variable specific growth rate fed-batch process, and glucose concentration should be controlled at a low level in l-tryptophan production, which suggested a combined feeding strategy of pseudo-exponential feeding and glucose-stat feeding for optimal l-tryptophan production. Applying this combined feeding strategy resulted in high cell density (54.5 g/L) and l-tryptophan production (38.8 g/L). In addition, a high glucose conversion rate (19.9%) with suitable ranges for specific growth rate and glucose concentration were obtained due to the low acetic acid concentration (0.9 g/L).

Effect of gene knockouts of l-tryptophan uptake system on the production of l-tryptophan in Escherichia coli

Uptake of l-tryptophan in Escherichia coli was carried out by three distinct permeases, Mtr, TnaB, and AroP, respectively. In the present study, the three genes of l-tryptophan uptake system were knocked out from an l-tryptophan-producing strain of E. coli, respectively. The knockout mutants all showed lower l-tryptophan uptake activities and higher l-tryptophan production than their parent. Among the three genes, the knockout of mtr was most critical for both l-tryptophan uptake and l-tryptophan production. The uptake activity of l-tryptophan of the mtr mutant was 1.5 nmol min −1 (mg dry weight) −1, which was decreased by 48% when compared to that of the parent; the production of l-tryptophan of the mtr mutant was 14.7 g/l, which was increased by 34% when compared to that of the parent. Furthermore, the physiological and fermentation characteristics caused by gene knockouts were also analyzed.

Development of l-tryptophan production strains by defined genetic modification in Escherichia coli

Construction and improvement of industrial strains play a central role in the commercial development of microbial fermentation processes. L-tryptophan producers have usually been developed by classical random mutagenesis due to its complicated metabolic network and regulatory mechanism. However, in the present study, an L-tryptophan overproducing Escherichia coli strain was developed by defined genetic modification methodology. Feedback inhibitions of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroF) and anthranilate synthase (TrpED) were eliminated by site-directed mutagenesis. Expression of deregulated AroF and TrpED was achieved by using a temperature-inducible expression plasmid pSV. Transcriptional regulation of trp repressor was removed by deleting trpR. Pathway for L-Trp degradation was removed by deleting tnaA. L-phenylalanine and L-tyrosine biosynthesis pathways that compete with L-tryptophan biosynthesis were blocked by deleting their critical genes (pheA and tyrA). The final engineered E. coli can produce 13.3 g/l of L-tryptophan. Fermentation characteristics of the engineered strains were also analyzed.

L-Tryptophan Production by Auxotrophic and Analogue Resistant Mutants of Aureobacterium flavescens

A number of tyrosine plus phenylalanine double auxotrophic mutants were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) treatment of a locally isolated strain of Aureobacterium flavescens of which 11A39 and 11A17 were selected on the basis of their tryptophan production in a mineral salt medium over other isolated mutant strains. The mutational block in the aromatic amino acid biosynthetic pathway of the selected double auxotrophs were determined. By controlling pH of the production medium to near neutrality, the active growth period could be extended up to 72 h and more tryptophan was accumulated compared to pH unregulated culture where the active growth ceased after 48 h. Further improvement of the tryptophan production has been achieved by stepwise isolation of a mutant strain resistant to the tryptophan analogues p-fluorotryptophan (FT) and 5-methyl tryptophan (MT) from the 11A39. Demand for L-tryptophan as food additive and therapeutic agent is increasing day by day throughout the World, particularly in the underdeveloped and developing countries like India. Still to date India depends on other countries for L-tryptophan. The aim of this work is to develop a potent high yielding, feed back insensitive mutant strain and optimization of its medium pH for maximum production of tryptophan.

Enzymatic synthesis of l-tryptophan from hair acid hydrolysis industries wastewater with tryptophan synthase

An effective method for production of L-tryptophan from hair acid hydrolysis wastewater (HHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative HHW utilization strategy. Tryptophan synthase could efficiently convert L-serine contained in HHW to L-tryptophan at pH 8.0, 40°C and Tween-80 of 0.04%. The enzyme also showed high tolerance to ammonium chloride, a component in HHW. In a scale up study, L-serine conversion rate reach 95.1% with a final L-tryptophan concentration of 33.2 g l(-1).

L-tryptophan production by Escherichia coli in the presence of Iranian cane molasses

The essential amino acid L-tryptophan (Trp) has importance as a pharmaceutical agent, especially in neuro-medicine. It is also added to feed products as a food fortifier. Furthermore, application of Trp is widespread in biotechnology. Trp is produced by a condensation reaction between indole and Lserine, which is catalyzed by bacterial tryptophanase activity. In this study, we have investigated Trp production using microbial system in the presence and absence of its precursors and Iranian cane molasses. The results showed that the optimum concentration of the molasses for maximum bacterial growth is 10 g/lit. Furthermore, in order to assay the amount of tryptophan produced, thin layer chromatography was used. The results showed that Iranian cane molasses contains considerable amounts of serine and indole, enough for Trp production (0.48 mM) in culture medium. But additional indole has inhibitory effects on Trp production. The data are compatible with previous reports on inhibitory effect of indole not only on cell growth but also on tryptophanase formation and function.

YddG fromEscherichia colipromotes export of aromatic amino acids

The inner membrane protein YddG of Escherichia coli is a homologue of the known amino acid exporters RhtA and YdeD. It was found that the yddG gene overexpression conferred resistance upon E. coli cells to the inhibiting concentrations of l-phenylalanine and aromatic amino acid analogues, dl-p-fluorophenylalanine, dl-o-fluorophenylalanine and dl-5-fluorotryptophan. In addition, yddG overexpression enhanced the production of l-phenylalanine, l-tyrosine or l-tryptophan by the respective E. coli-producing strains. On the other hand, the inactivation of yddG decreased the aromatic amino acid accumulation by these strains. The cells of the E. colil-phenylalanine-producing strain containing overexpressed yddG accumulated less l-phenylalanine inside and exported the amino acid at a higher rate than the cells of the isogenic strain containing wild-type yddG. Taken together, these results indicate that YddG functions as an aromatic amino acid exporter.