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Modification of tryptophan transport system and its impact on production of l -tryptophan in Escherichia coli
Bioresource Technology 114 (2012) 549–554
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Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli Qian Liu, Yongsong Cheng, Xixian Xie, Qingyang Xu, Ning Chen ⇑ College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin 300457, China
a r t i c l e
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Article history: Received 14 November 2011 Received in revised form 16 February 2012 Accepted 17 February 2012 Available online 10 March 2012 Keywords: Escherichia coli Tryptophan transport system yddG aroP Fermentation of L-tryptophan
a b s t r a c t 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 DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-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 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction L-tryptophan as the third limiting amino acid is widely used as the common feedstocks (Hinman, 1991). Due to its commercial importance, a number of researchers are being devoted to explore L-tryptophan production. The traditional methods for the production of L-tryptophan are composed of chemical synthesis, direct fermentation bioconversion, and enzymatic conversion of precursors (Leuchtenberger et al., 2005; Aiba et al., 1980; Tribe and Pittard, 1979; Zhao et al., 2010). Microbial fermentation allows the production of L-tryptophan from cheap and renewable carbon source such as sucrose or glucose (Sprenger, 2007) and, therefore it is usually more favorable than biotransformation processes. In normal conditions, the strategy of L-tryptophan-producing strains requires first alleviation of all control levels in the biosynthesis pathway, such as repression, attenuation and feedback, which made it difficult to redirect the carbon flux towards L-tryptophan (Ikeda and Katsumata, 1994), to identify and remove rate-limiting steps by the appropriate overproduction of enzymes of the general aromatic amino acid pathway (Dell and Frost, 1993; Ikeda, 2003), and then to reduce competing pathways and to improve and balance precursor supply both in the common pathway as well as in the specific branch (Ikeda, 2003, 2006). However, the biosyn-
⇑ Corresponding author. Address: Metabolic Engineering Laboratory, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin 300457, China. Tel.: +86 22 60601251; fax: +86 22 60602198. E-mail address: [email protected] (N. Chen). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.02.088
thesis pathway of L-tryptophan is subjected to multiple regulations at several steps (Berry, 1996), thus it is not easy to remove completely all regulatory controls existing in the pathway. In recent years, a new type of producer with a different production mechanism has been developed in which feedback control does not operate when an amino acid is overproduced because of the low level of intracellular amino acid concentration. Some of these strains with altered transport systems are distinguished from classical regulatory mutants. The well-known examples are the secretion of glutamate and threonine by Corynebacterium glutamicum and Escherichia coli, respectively (Gourdon and Lindley, 1999; Clément and Lanéelle, 1986; Livshits et al., 2003; Gosset, 2009). In the biosynthesis of L-tryptophan, on the one hand, transport and re-uptake of products and cause unwanted futile cycles (Krämer, 1994b); on the other hand, efflux of L-tryptophan from producing cells is generally assumed to proceed via simple diffusion as its hydrophobicity, while an involvement of excretion mechanisms cannot be ruled out yet. Therefore, more detailed studies of the transport systems of L-tryptophan are needed. It has been reported that E. coli possesses three permeases including AroP, TnaB, and Mtr, which play different roles in accumulation of intracellular tryptophan (Yanofsky et al., 1991), as shown in supporting information (SI) Fig. 1 Fig. 1. AroP, a general aromatic amino acid permease, encoding the aroP gene, can transport phenylalanine and tyrosine with high affinity (Brown, 1970; Honoré and Cole, 1990; Sarsero et al., 1991). However, Mtr and TnaB are specific for L-tryptophan. TnaB is a low-affinity transporter encoded in the tryptophanase operon together with the tnaA gene (Koyanagi et al., 2004); Mtr, a high-affinity tryptophan
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15 g, (NH4)2SO4 10 g, sodium citrate 0.5 g, MgSO47H2O 5 g, KH2PO4 1.5 g, FeSO47H2O 15 mg, VB1 100 mg. Fermentation medium (per liter) was composed of glucose 10 g, yeast extract 1 g, (NH4)2SO4 4 g, sodium citrate 2 g, MgSO47H2O 5 g, KH2PO4 2 g, FeSO47H2O 100 mg. When required, antibiotics were added as follows: ampicillin (AmR), 100 lg/mL; chloramphenicol (CmR), 30 lg/ mL; tetracycline (TetR), 50 lg/mL.
Glucose PEP+Ery4P DAHP SHIK Ar oP
Tryptophan
CHA
Mtr
ANTA
TnaB
Tryptophan
Catabolism
YddG
Anabolism
Fig. 1. Biosynthesis pathways and transport systems of tryptophan in E. coli. DAHP: 3-deoxy-D-arobino-hetulosonate-7-phosphate; SHIK: shikimic acid; CHA: chorismic acid; ANTA: anthranilate.
permease, also can transport indole (Yanofsky et al., 1991). Three tryptophan transporters that are secondary carriers belong to amino acid/polyamine/organocation (APC) superfamily (TC (transporter classification) No. 2.A.3) (Jack et al., 2000; Milner et al., 1987; http://www.tcdb.org/). The genes for these permeases have been cloned and sequenced (Honoré and Cole, 1990; Heatwole and Somerville, 1991; Edwards and Yudkin, 1982). Due to the hydrophobic properties, aromatic amino acids can effectively cross the bacterial cell membrane (Krämer, 1994b). The researchers are doubtful for proteins involved in the specific efflux of aromatic amino acids from cells. Whether the overexpression of yddG can enhance the production of L-phenylalanine, L-tyrosine, and L-tryptophan by E. coli-producing strains is still debated (Doroshenko et al., 2007). Final result indicates that YddG can function as an aromatic amino acid exporter (SI Fig. 1). YddG is classified as aromatic amino acid/paraquat/exporter (ArAA/P-E) family (TC No. 2.A.7.17) of the drug/metabolite transporter (DMT) (TC No. 2.A.7) superfamily of protein (Airich et al., 2010). Due to the lack of relevant research in the transport systems of L-tryptophan, the role of transport systems in fermentation of L-tryptophan has not been explored in industry. The objective of this work was to describe the effects on the growth of L-tryptophan-producing strain, E. coli TRTH, with several modifications in transport systems (export and import) of L-tryptophan and the production of L-tryptophan. In order to accomplish this aim, three recombinant strains such as E. coli TRTH DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-Y have been described by flask-shaking fermentation and fermentation in a 30-liter fermentor. 2. Methods 2.1. Bacterial strains, primers and plasmids E. coli strains, primers and plasmids used in this study were listed in Table 1. 2.2. Media and culture conditions LB, SOB, SOC, and M9 minimal media were prepared as previous description (Ausubel et al., 1995). The 2-YT medium (per liter) was composed of tryptone 16 g, yeast extract 10 g and NaCl 5 g. Seed medium (per liter) was composed of glucose 20 g, yeast extract
(i) Tube-test. Isolated colonies of test strains were selected to cultivate in 5 mL of LB medium with shaking at 37 °C for 12 h. (ii) Shaking flasks. Two milliliter of seed culture at OD660 of 0.4 at 32 °C in seed medium was inoculated into 500 mL shaking flask containing 20 mL of fermentation medium. The pH of the medium was maintained at about 6.4 with NH4OH. Cultivation of all strains was cultivated under the presence of tetracycline or/and chloramphenicol. (iii) Fermentor. The cells were pre-cultured at 32 °C for 14 h in 5-liter fermentor containing 3 L of seed medium plus tetracycline or/and chloramphenicol. 1.6 L of pre-culture was transported into the 30-liter fermentor containing 16 L of fermentation medium plus tetracycline or/and chloramphenicol. The pH was controlled automatically at 6.4 with NH4OH. 2.3. Construction of the strains Gene aroP disruption in the chromosome of E. coli K-12 was obtained by Red helper plasmid, pKD46 (Datsenko and Wanner, 2000). The linear DNA fragment was obtained by polymerase chain reaction (PCR) using primers P1 and P2 with helper plasmid pKD3. In order to eliminate the CmR gene from the integrated locus, the cells were transformed with plasmid pCP20 carrying the FLP recombinase gene. All test PCRs used primers P3 and P4 exhibited that all mutants had correct structures. 2.4. Development of recombinant plasmid pSTV28-Y The gene yddG was amplified by PCR with primers P5 and P6, with the chromosome of E. coli K-12 MG1655 as the template. The PCR product was purified using the purification kit. Plasmid DNA was isolated according to the alkaline lysis and purified by CsCl-ethidium bromide equilibrium centrifugation. The purified yddG fragment was digested with EcoRI and BamHI to obtain a 939 bp DNA fragment. The newly achieved DNA fragment was then inserted into pSTV28 to construct a plasmid, pSTV28-Y. 2.5. Assay of intracellular L-tryptophan concentration Intracellular metabolites should be exposed to multifarious analytical procedures, usually by exposing cells to cell membrane permeabilizing agents. The agents for extracting intracellular metabolites of E. coli included boiling 75% ethanol (v/v), 50–100% methanol, perchloric acid and KOH (Mashego et al., 2007). Considering the solubility of L-tryptophan, the analysis for intracellular concentration of L-tryptophan adopted the procedures developed by Rabinowitz and Kimball (2007) and Doroshenko et al. (2007), with some slight modifications. The cells of tested strains, E. coli TRTH, E. coli TRTH DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-Y, were grown in M9 minimal medium at 37 °C with vigorous shaking until the OD600 of 2.0. Then, the cells were washed with 0.9% NaCl and concentrated twice with M9 minimal medium. Aliquots were sampled at the designated time intervals. Totally 8 mL sample was centrifuged at 2500g for 5 min. The cell pellet was washed twice with 0.9% NaCl. Then 500 lL of 80% (v/v) acetonitrile with 0.1 M formic acid was added to the cell pellet to initiate the
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Q. Liu et al. / Bioresource Technology 114 (2012) 549–554 Table 1 Strains, plasmids and primers used in this study. Name
Characteristics
Source
Strains E. coli K-12
Wild type, MG1655
trpEDCBA+TetR, DtnaA, DaroP trpEDCBA+TetR, yddG+CmR, DtnaA trpEDCBA + TetR, yddG + CmR, DtnaA, DaroP yddG + CmR aroP + CmR
Center of Industrial Culture Collection of Tianjin University of Science and Technology Center of Industrial Culture Collection of Tianjin University of Science and Technology This study This study This study This study This study
Plasmids pKD46 pKD3 pCP20 pSTV28
AmR, k Red-expressing vector CmR, Template vector AmR, CmR, FLP-expressing vector CmR, cloning vector
In this laboratory In this laboratory In this laboratory TaKaRa
Primersa P1
50 -AAACTTACACACGCATCACTGCGTAGATCAAAAAAACAACC
This study
E. coli TRTH E. E. E. E. E.
coli coli coli coli coli
TRTH DaroP TRTH-Y TRTH DaroP-Y K-12-Y K-12-A
R
trpEDCBA+Tet , DtnaA
ACCGCACGAGGTTTCTTGAGCGATTGTGTAGGCTGGAG-30 P2 P3 P4 P5 P6 a
50 -GGGCGTTGGTGTAAAGATTATTGCCCTCACCCTGTACGGG TGAGGGCGTAGAGAGATAACGGCTGACATGGGAATTAGC-30 50 -CTTGATCTGACGGAAGTCTTTTTG-30 50 -TGGTGATTGCACTACTGACGATTTAC-30
This study
50 -CTCGGCGAATTCCGGCAAGAGAGACAAAACAG-30 (EcoRI)
This study This study This study
50 -TAGTCAGGATCCCCGTAGACCCGGCAGTTAT-30 (BamHI)
This study
The first 56 nucleotides in P1 and P2 sequences (underlined) correspond to the sequence in the upper and lower strands of the aroP gene.
extraction process at 4 °C. After incubation for 15 min, the mixture was frozen at 80 °C for 15 min. Then, the suspension was centrifuged at 2500g for 5 min at 4 °C to collect the supernatant. The resulting pellet was then re-extracted once. All supernatants were frozen in liquid nitrogen, freeze-dried, and stored at 80 °C for further analysis, and the sample was re-suspended with 400 lL of water. In order to detect amino acids, the samples were analyzed by high-performance liquid chromatography. 2.6. Disk diffusion test Before plating, the strains were grown in minimal medium at 37 °C with shaking to stationary phase. Then, the cultures were washed with 0.9% NaCl and concentrated for 10 times and 0.1 mL of these suspensions was placed on the top of the minimal medium plates. The analog solutions of different concentrations were applied to the disk. The plates were cultivated at 37 °C for 36 h. 2.7. Analysis L-tryptophan titer, and glucose concentration were determined as described previously (Cheng et al., 2010). Cell dry weight was determined by centrifuging the sample, washing cell pellet twice with 0.9% NaCl, and drying it at 90 °C for 24 h. Cell growth was monitored spectrophotometrically by measurement of the optical density.
Table 2. The sensitivity of 5-MT was removed by E. coli K-12-Y, suggesting that the protein YddG expressed from pSTV28-Y was activated. However, the L-tryptophan-producing strains with feedback inhibition were not highly sensitive to 5-MT. Therefore, in order to provide the successful construction of E. coli TRTH DaroP, the test PCR was necessary. 3.2. Effect of transport systems in L-tryptophan-producing strains on the accumulation of L-tryptophan The cells grown to the middle of the logarithmic phase were collected and tested in a short-term fermentation in M9 minimal medium and the intracellular tryptophan was extracted by acidic acetonitrile as described previously. The concentrations of extracellular and intracellular tryptophan were demonstrated in Fig. 2. The concentration of L-tryptophan in medium was shown in Fig. 2(a), and the recombinant strains revealed a higher concentration of extracellular tryptophan than that by strain E. coli TRTH at 6 h, and one-way ANOVA with Tukey’s test at the significance level of 0.05 proved that the results were significantly different (F > Fcrit). However, the result also indicated a higher concentration of extracellular tryptophan by E. coli TRTH DaroP than by other recombinant strains and their parent during the early phase of shortterm fermentation (<3 h). On the one hand, in the medium lacking of L-phenylalanine and L-tyrosine, the AroP permease was activated in tryptophan transport (Yanofsky et al., 1991). Therefore, in the
3. Results and discussion 3.1. Construction of recombinant plasmid and strain Many aromatic amino acid analogs, such as 5-methyltryptophan (5-MT), were transported by transport systems for aromatic amino acids, such as AroP and YddG (Kuhn, 1977; Tobias, 2010). For this reason, the bioactivity of protein YddG, expressed from recombinant plasmid pSTV28-Y, could be verified by assaying the sensitivity of 5-MT. Disk diffusion test results were presented in
Table 2 Disk diffusion test on 5-MT. Strain
E. coli K-12 E. coli K-12-Y a
Zone of inhibition (cm) of straina The concentration of 5-MT 15 lg
5 lg
3.8 <1
2.8 <1
The diameter of growth inhibition in centimeters.
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Fig. 2. Concentration of tryptophan in M9 minimal medium (a) and cytoplasma (b). The sample was collected once an hour. The extraction method of intracellular tryptophan was adopted the procedure described by Rabinowitz and Kimball (2007). The concentration of tryptophan was determined by HPLC.
M9 minimal medium, a very small amount of extracellular tryptophan could be recaptured to the cytoplasm in the case of the disruption of gene aroP. On the other hand, at the preliminary stage of cell growth, due to the limited amino acids, such as the aromatic amino acids, the participation in the biosynthesis of proteins, the biosynthesis of protein YddG and other proteins could be prevented, while E. coli TRTH-Y and E. coli TRTH DaroP-Y were required to synthesize amount of carrier protein, YddG. In this case, because of the limited biosynthesis of YddG, its ability to export L-tryptophan could be limited, as characterized by less accumulation of L-tryptophan in medium. However, as the increase in the short-term fermentation, the ability to produce aromatic amino acids by strains was improved and characterized by an increase of expression level of yddG. In this case, the bio-function of carrier protein YddG was enhanced. Therefore, more endogenetic tryptophan could be exported to the medium. Conventionally, in the fermentation process of amino acid production, some strains with altered transport systems have been used for industrial production. These strains were aimed primarily at the deregulation of the relevant biosynthetic pathway so that feedback inhibition did not work when an excess amount of a desired amino acid was intracellularly accumulated. As shown in Fig. 2(b), a lower level of endogenetic tryptophan in E. coli TRTH than in recombinant strains was observed. Krämer and his co-workers (Krämer, 1994a) have reported that three different models were developed to explain amino acid excretion: overflow metabolism, limited catabolism, and deregulated anabolism. Glutamate production in C. glutamicum was considered as a typical metabolic overflow situation. Similarly, in this study, the increasing concentration of intracellular tryptophan could be explanted as the enhanced production capacity of L-tryptophan, causing by some reasons, such as the redistribution of metabolic flow. For the strain E. coli TRTH DaroP, the amount of L-tryptophan recaptured from medium could be reduced, caused by the disruption of gene aroP. In addition, based on the fact that efflux systems, such as exporter YddG, seem to be expressed at a very low level (Doroshenko et al., 2007), L-tryptophan produced continually in the cytoplasm could not be exported to medium timely. In this case, E. coli TRTH DaroP should reveal a higher concentration of intracellular tryptophan than E. coli TRTH. Although the expression level of yddG of strains, E. coli TRTH-Y and E. coli TRTH DaroP-Y, was much higher than that of E. coli TRTH and E. coli TRTH DaroP, because of the saturation of exporter YddG, the transport capacity of YddG should probably be inactive to reduce the concentration of intracellular tryptophan. In conclusion, the modifications of transport system could result in a greater production of L-tryptophan by some reason, such
as the metabolic flux redistribution of L-tryptophan biosynthesis pathway. 3.3. Parameters of L-tryptophan-producing strains by flask-shaking fermentation Large volume of proteins and enzymes were required during the phase of cell growth and amino acid production, and the externally available amino acids, in the cytoplasm, was used directly for protein synthesis without energy consumption for anabolism (Burkovski and Krämer, 2002). In this case, the amount of the externally available amino acids was pivotal for protein synthesis, and had significant effects on biomass. The biomass of each strain by flask-shaking was demonstrated in Fig. 3. Based on the previously published report, the aromatic amino acid pools formed by AroP were the major components to participate in protein synthesis (Tobias, 2010). Therefore, the apparently lower biomass in the aroP disruption background indicated that the deficiency of aroP could be disadvantageous to cell growth due to the protein deficiency resulted from participated aromatic amino acids, as shown in Fig. 3. In the present study, the over-expression of gene yddG in L-tryptophan-producing strains was not associated with its impact on cell growth, as shown in the Fig. 3. However, over-expression of gene yddG in the wild strain showed a different result with L-tryptophan-producing strain. In the tube-fermentation, the biomass of E. coli K-12-Y (OD600 = 3.20 ± 0.06), was much lower than that of E. coli K-12 (OD600 = 3.95 ± 0.03) and E. coli K-12 carrying pSTV28 (OD600 = 3.80 ± 0.04), which indicated that over-expression of gene yddG had an adverse impact on cell growth of wild strain. The possible explanation for this phenomenon maybe the different demands of aromatic amino acids during the initial stage of cell growth. As an aromatic amino acid exporter, the over-expression of YddG resulted in a high efflux of aromatic amino acids, maintaining the normal growth of wild strains. While for the producing strains, the biosynthesis of aromatic amino acids by them was enough to satisfy the requirement of aromatic amino acids during the phase of cell growth and L-tryptophan production. In the flask-shaking fermentation, the glucose conversion rates of E. coli TRTH DaroP and E. coli TRTH-Y were 5.2% and 6.2%, respectively, and higher than their parent (4.4%), which suggested that the modification of transport system could increase the metabolic flux of L-tryptophan biosynthesis pathway. Ordinary modification characterized by over-expression of the export and inactivation of the uptake can be beneficial to the deregulating feedback inhibition during the fermentation phase, and improving the production of amino acids. However, the glucose conversion rate of E. coli
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1.8
40
1.6
35
Production of tryptophan g/l
1.4 1.2
OD660
1.0 0.8 0.6 E. coli TRTH E. coli aroP-Y E. coli TRTH-Y aroP E. coli
0.4 0.2 0.0 0
5
10
15
20
25
30
35
40
Time h Fig. 3. Cell growth curve in flask-shaking fermentation. The curve of biomass by flask shaking: the samples were diluted 1:20 with deionized water to determine the absorbance at 660 nm.
TRTH DaroP-Y was 4.6%, which did not reveal an increase as expectation, in this study. The possible reason could be due to the limited endogenetic aromatic amino acids participated in the proteins biosynthesis because this strain exported intracellular aromatic amino acids effectively and imported limited amount of extracellular amino acids to the cytoplasm. In this case, aromatic amino acids synthesized by glucose could participate in the protein synthesis rather than be exported to the medium. However, E. coli TRTH DaroP and E. coli TRTH-Y were protected from this phenomenon, which is possibly due to the fact that the concentration of aromatic amino acids participating in synthesis of proteins in E. coli TRTH DaroP and E. coli TRTH-Y cannot decrease too low to inhibit the biosynthesis of proteins. In contrast, the concentration of extracellular tryptophan in E. coli TRTH DaroP-Y in M9 minimal medium was the highest as shown in Fig. 2(a), which was different from the case in the fermentation medium. The most likely explanations for this discrepancy were shown as below. The non-growing cells in the minimal medium required less aromatic amino acids to satisfy cell growth and more to flow to the medium. At the meanwhile, in E. coli TRTH DaroP-Y, more endogenetic aromatic amino acids were exported to the medium and less extracellular aromatic amino acids were imported to the inner part of the cells These two reasons resulted in the fact that the extracellular concentration in E. coli TRTH DaroP-Y was higher when cultured in the minimal medium. In conclusion, based on the results in shaking-flask fermentation and the comparison on the glucose conversion rates of the test strains, the moderate modifications of transport system resulted in the redistribution of L-tryptophan metabolic flux, and had an effect on the cell growths.
30 25 20 15 10 5
E. coli TRTH-Y E. coli TRTH
0 -5 0
5
10
15
20
25
30
35
40
Time h Fig. 4. Production curve of tryptophan fermented by E. coli TRTH and E. coli TRTH-Y.
other hand, the result could be explained by the fact that the intrinsic transport system of E. coli has satisfied the production of L-tryptophan. Finally, in the L-tryptophan-producing strains, the disadvantage caused by increasing intracellular tryptophan concentration was not obvious. In addition, the L-tryptophan production rate, the glucose consumption rate, and biomass of strains revealed an obvious decrease at 36 h. Therefore, the fermentation was terminated at 36 h. 4. Conclusion In this study, the conclusions were proved that modifications of transport systems for L-tryptophan resulted in a greater production of L-tryptophan by the metabolic flux redistribution of L-tryptophan biosynthesis partway, and strengthen of exporter was more effective than other modifications in the fermentation of L-tryptophan, and the final output in the 30-liter fermentor was 36.3 g/L, which increased by 12.6% compared with that by E. coli TRTH. Acknowledgements This work was supported by program for Changjiang Scholars and Innovative Research Team in University (IRT1166). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012. 02.088. References
3.4. Fed-batch fermentation of E. coli TRTH-Y in 30-liter fermentor Because of relatively higher glucose conversion rate in flaskshaking fermentation, fed-batch fermentation of E. coli TRTH-Y was carried out in 30-liter fermentor to explore the production of L-tryptophan. After the fermentation for 36 h, L-tryptophan production by recombinant strain was 36.3 g/L, which increased by 12.6% compared with that by E. coli TRTH, as shown in Fig. 4. However, the result presented in the 30-liter fermentation did not exhibit a significant improvement in the production of L-tryptophan by recombinant strains. On the one hand, the composition of medium could affect the capacity of transport systems. On the
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Krämer, R., 1994a. Secretion of amino acids by bacteria: physiology and mechanism. FEMS Microbiol. Rev. 13, 75–93. Krämer, R., 1994b. Systems and mechanisms of amino acid uptake and excretion in prokaryotes. Arch. Microbiol. 162, 1–13. Koyanagi, T., Katayama, T., Suzuki, H., Kumagai, H., 2004. Identification of the LIV-I/ LS system as the third phenylalanine transporter in Escherichia coli. J. Biotechnol. 186, 343–350. Kuhn, J., 1977. Detection of antimetabolite activity: effects and transport of tryptophan analogs in Escherichia coli. Antimicrob. Agents Chemother. 12, 322– 327. Leuchtenberger, W., Huthmacher, K., Drauz, K., 2005. Biotechnological production of amino acids and derivatives: current status and prospects. Appl. Microbiol. Biotechnol. 69, 1–8. Livshits, V.A., Zakataeva, N.P., Aleshin, V.V., Vitushkina, M.V., 2003. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. Res. Microbiol. 154, 123–135. Milner, J.L., Vink, B., Wood, J.M., 1987. Transmembrane amino acid flux in bacterial cells. Crit. Rev. Biotechnol. 5, 1–47. Mashego, M.R., Rumbold, K., De Mey, M., Vandamme, E., Soetaert, W., Heijnen, J.J., 2007. Microbial metabolomics: past, present and future methodologies. Biotechnol. Lett. 29, 1–16. Rabinowitz, J.D., Kimball, E., 2007. Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal. Chem. 79, 6167–6173. Sarsero, J.P., Wookey, P.J., Gollnick, P., Yanofsky, C., Pittard, A.J., 1991. A new family of integral membrane proteins involved in transport of aromatic amino acids in Escherichia coli. J. Bacteriol. 173, 3231–3234. Sprenger, G.A., 2007. Aromatic amino acids. In: Wendisch, V.F. (Ed.), Amino Acid Biosynthesis-Pathways, Regulation and Metabolic Engineering (Microbiology Monographs, vol 5). Springer-Verlag, Berlin Heidelberg, pp. 103–127. Tobias, M., 2010. Untersuchungen zum Export von Tryptophan in Escherichia coli. PhD Thesis. Universität zu Köln, Mathematisch-Naturwissenschaftlichen Fakultät. Tribe, D.E., Pittard, J., 1979. Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation. Appl. Environ. Microbiol. 38, 181–190. Yanofsky, C., Horn, V., Gollnick, P., 1991. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli. J. Bacteriol. 173, 6009–6017. Zhao, G.H., Liu, J.Z., Dong, K., Zhang, F., Zhang, H.G., Liu, Q., Jiao, Q.C., 2010. Enzymatic synthesis of L-tryptophan from hair acid hydrolysis industries wastewater with tryptophan synthase. Bioresour. Technol. 102, 3554–3557.
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Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli Qian Liu, Yongsong Cheng, Xixian Xie, Qingyang Xu, Ning Chen ⇑ College of Biotechnology, Tianjin University of Science and Technology, Key Laboratory of Industrial Microbiology of Education Ministry, Tianjin 300457, China
a r t i c l e
i n f o
Article history: Received 14 November 2011 Received in revised form 16 February 2012 Accepted 17 February 2012 Available online 10 March 2012 Keywords: Escherichia coli Tryptophan transport system yddG aroP Fermentation of L-tryptophan
a b s t r a c t 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 DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-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 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction L-tryptophan as the third limiting amino acid is widely used as the common feedstocks (Hinman, 1991). Due to its commercial importance, a number of researchers are being devoted to explore L-tryptophan production. The traditional methods for the production of L-tryptophan are composed of chemical synthesis, direct fermentation bioconversion, and enzymatic conversion of precursors (Leuchtenberger et al., 2005; Aiba et al., 1980; Tribe and Pittard, 1979; Zhao et al., 2010). Microbial fermentation allows the production of L-tryptophan from cheap and renewable carbon source such as sucrose or glucose (Sprenger, 2007) and, therefore it is usually more favorable than biotransformation processes. In normal conditions, the strategy of L-tryptophan-producing strains requires first alleviation of all control levels in the biosynthesis pathway, such as repression, attenuation and feedback, which made it difficult to redirect the carbon flux towards L-tryptophan (Ikeda and Katsumata, 1994), to identify and remove rate-limiting steps by the appropriate overproduction of enzymes of the general aromatic amino acid pathway (Dell and Frost, 1993; Ikeda, 2003), and then to reduce competing pathways and to improve and balance precursor supply both in the common pathway as well as in the specific branch (Ikeda, 2003, 2006). However, the biosyn-
⇑ Corresponding author. Address: Metabolic Engineering Laboratory, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin 300457, China. Tel.: +86 22 60601251; fax: +86 22 60602198. E-mail address: [email protected] (N. Chen). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.02.088
thesis pathway of L-tryptophan is subjected to multiple regulations at several steps (Berry, 1996), thus it is not easy to remove completely all regulatory controls existing in the pathway. In recent years, a new type of producer with a different production mechanism has been developed in which feedback control does not operate when an amino acid is overproduced because of the low level of intracellular amino acid concentration. Some of these strains with altered transport systems are distinguished from classical regulatory mutants. The well-known examples are the secretion of glutamate and threonine by Corynebacterium glutamicum and Escherichia coli, respectively (Gourdon and Lindley, 1999; Clément and Lanéelle, 1986; Livshits et al., 2003; Gosset, 2009). In the biosynthesis of L-tryptophan, on the one hand, transport and re-uptake of products and cause unwanted futile cycles (Krämer, 1994b); on the other hand, efflux of L-tryptophan from producing cells is generally assumed to proceed via simple diffusion as its hydrophobicity, while an involvement of excretion mechanisms cannot be ruled out yet. Therefore, more detailed studies of the transport systems of L-tryptophan are needed. It has been reported that E. coli possesses three permeases including AroP, TnaB, and Mtr, which play different roles in accumulation of intracellular tryptophan (Yanofsky et al., 1991), as shown in supporting information (SI) Fig. 1 Fig. 1. AroP, a general aromatic amino acid permease, encoding the aroP gene, can transport phenylalanine and tyrosine with high affinity (Brown, 1970; Honoré and Cole, 1990; Sarsero et al., 1991). However, Mtr and TnaB are specific for L-tryptophan. TnaB is a low-affinity transporter encoded in the tryptophanase operon together with the tnaA gene (Koyanagi et al., 2004); Mtr, a high-affinity tryptophan
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15 g, (NH4)2SO4 10 g, sodium citrate 0.5 g, MgSO47H2O 5 g, KH2PO4 1.5 g, FeSO47H2O 15 mg, VB1 100 mg. Fermentation medium (per liter) was composed of glucose 10 g, yeast extract 1 g, (NH4)2SO4 4 g, sodium citrate 2 g, MgSO47H2O 5 g, KH2PO4 2 g, FeSO47H2O 100 mg. When required, antibiotics were added as follows: ampicillin (AmR), 100 lg/mL; chloramphenicol (CmR), 30 lg/ mL; tetracycline (TetR), 50 lg/mL.
Glucose PEP+Ery4P DAHP SHIK Ar oP
Tryptophan
CHA
Mtr
ANTA
TnaB
Tryptophan
Catabolism
YddG
Anabolism
Fig. 1. Biosynthesis pathways and transport systems of tryptophan in E. coli. DAHP: 3-deoxy-D-arobino-hetulosonate-7-phosphate; SHIK: shikimic acid; CHA: chorismic acid; ANTA: anthranilate.
permease, also can transport indole (Yanofsky et al., 1991). Three tryptophan transporters that are secondary carriers belong to amino acid/polyamine/organocation (APC) superfamily (TC (transporter classification) No. 2.A.3) (Jack et al., 2000; Milner et al., 1987; http://www.tcdb.org/). The genes for these permeases have been cloned and sequenced (Honoré and Cole, 1990; Heatwole and Somerville, 1991; Edwards and Yudkin, 1982). Due to the hydrophobic properties, aromatic amino acids can effectively cross the bacterial cell membrane (Krämer, 1994b). The researchers are doubtful for proteins involved in the specific efflux of aromatic amino acids from cells. Whether the overexpression of yddG can enhance the production of L-phenylalanine, L-tyrosine, and L-tryptophan by E. coli-producing strains is still debated (Doroshenko et al., 2007). Final result indicates that YddG can function as an aromatic amino acid exporter (SI Fig. 1). YddG is classified as aromatic amino acid/paraquat/exporter (ArAA/P-E) family (TC No. 2.A.7.17) of the drug/metabolite transporter (DMT) (TC No. 2.A.7) superfamily of protein (Airich et al., 2010). Due to the lack of relevant research in the transport systems of L-tryptophan, the role of transport systems in fermentation of L-tryptophan has not been explored in industry. The objective of this work was to describe the effects on the growth of L-tryptophan-producing strain, E. coli TRTH, with several modifications in transport systems (export and import) of L-tryptophan and the production of L-tryptophan. In order to accomplish this aim, three recombinant strains such as E. coli TRTH DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-Y have been described by flask-shaking fermentation and fermentation in a 30-liter fermentor. 2. Methods 2.1. Bacterial strains, primers and plasmids E. coli strains, primers and plasmids used in this study were listed in Table 1. 2.2. Media and culture conditions LB, SOB, SOC, and M9 minimal media were prepared as previous description (Ausubel et al., 1995). The 2-YT medium (per liter) was composed of tryptone 16 g, yeast extract 10 g and NaCl 5 g. Seed medium (per liter) was composed of glucose 20 g, yeast extract
(i) Tube-test. Isolated colonies of test strains were selected to cultivate in 5 mL of LB medium with shaking at 37 °C for 12 h. (ii) Shaking flasks. Two milliliter of seed culture at OD660 of 0.4 at 32 °C in seed medium was inoculated into 500 mL shaking flask containing 20 mL of fermentation medium. The pH of the medium was maintained at about 6.4 with NH4OH. Cultivation of all strains was cultivated under the presence of tetracycline or/and chloramphenicol. (iii) Fermentor. The cells were pre-cultured at 32 °C for 14 h in 5-liter fermentor containing 3 L of seed medium plus tetracycline or/and chloramphenicol. 1.6 L of pre-culture was transported into the 30-liter fermentor containing 16 L of fermentation medium plus tetracycline or/and chloramphenicol. The pH was controlled automatically at 6.4 with NH4OH. 2.3. Construction of the strains Gene aroP disruption in the chromosome of E. coli K-12 was obtained by Red helper plasmid, pKD46 (Datsenko and Wanner, 2000). The linear DNA fragment was obtained by polymerase chain reaction (PCR) using primers P1 and P2 with helper plasmid pKD3. In order to eliminate the CmR gene from the integrated locus, the cells were transformed with plasmid pCP20 carrying the FLP recombinase gene. All test PCRs used primers P3 and P4 exhibited that all mutants had correct structures. 2.4. Development of recombinant plasmid pSTV28-Y The gene yddG was amplified by PCR with primers P5 and P6, with the chromosome of E. coli K-12 MG1655 as the template. The PCR product was purified using the purification kit. Plasmid DNA was isolated according to the alkaline lysis and purified by CsCl-ethidium bromide equilibrium centrifugation. The purified yddG fragment was digested with EcoRI and BamHI to obtain a 939 bp DNA fragment. The newly achieved DNA fragment was then inserted into pSTV28 to construct a plasmid, pSTV28-Y. 2.5. Assay of intracellular L-tryptophan concentration Intracellular metabolites should be exposed to multifarious analytical procedures, usually by exposing cells to cell membrane permeabilizing agents. The agents for extracting intracellular metabolites of E. coli included boiling 75% ethanol (v/v), 50–100% methanol, perchloric acid and KOH (Mashego et al., 2007). Considering the solubility of L-tryptophan, the analysis for intracellular concentration of L-tryptophan adopted the procedures developed by Rabinowitz and Kimball (2007) and Doroshenko et al. (2007), with some slight modifications. The cells of tested strains, E. coli TRTH, E. coli TRTH DaroP, E. coli TRTH-Y, and E. coli TRTH DaroP-Y, were grown in M9 minimal medium at 37 °C with vigorous shaking until the OD600 of 2.0. Then, the cells were washed with 0.9% NaCl and concentrated twice with M9 minimal medium. Aliquots were sampled at the designated time intervals. Totally 8 mL sample was centrifuged at 2500g for 5 min. The cell pellet was washed twice with 0.9% NaCl. Then 500 lL of 80% (v/v) acetonitrile with 0.1 M formic acid was added to the cell pellet to initiate the
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Characteristics
Source
Strains E. coli K-12
Wild type, MG1655
trpEDCBA+TetR, DtnaA, DaroP trpEDCBA+TetR, yddG+CmR, DtnaA trpEDCBA + TetR, yddG + CmR, DtnaA, DaroP yddG + CmR aroP + CmR
Center of Industrial Culture Collection of Tianjin University of Science and Technology Center of Industrial Culture Collection of Tianjin University of Science and Technology This study This study This study This study This study
Plasmids pKD46 pKD3 pCP20 pSTV28
AmR, k Red-expressing vector CmR, Template vector AmR, CmR, FLP-expressing vector CmR, cloning vector
In this laboratory In this laboratory In this laboratory TaKaRa
Primersa P1
50 -AAACTTACACACGCATCACTGCGTAGATCAAAAAAACAACC
This study
E. coli TRTH E. E. E. E. E.
coli coli coli coli coli
TRTH DaroP TRTH-Y TRTH DaroP-Y K-12-Y K-12-A
R
trpEDCBA+Tet , DtnaA
ACCGCACGAGGTTTCTTGAGCGATTGTGTAGGCTGGAG-30 P2 P3 P4 P5 P6 a
50 -GGGCGTTGGTGTAAAGATTATTGCCCTCACCCTGTACGGG TGAGGGCGTAGAGAGATAACGGCTGACATGGGAATTAGC-30 50 -CTTGATCTGACGGAAGTCTTTTTG-30 50 -TGGTGATTGCACTACTGACGATTTAC-30
This study
50 -CTCGGCGAATTCCGGCAAGAGAGACAAAACAG-30 (EcoRI)
This study This study This study
50 -TAGTCAGGATCCCCGTAGACCCGGCAGTTAT-30 (BamHI)
This study
The first 56 nucleotides in P1 and P2 sequences (underlined) correspond to the sequence in the upper and lower strands of the aroP gene.
extraction process at 4 °C. After incubation for 15 min, the mixture was frozen at 80 °C for 15 min. Then, the suspension was centrifuged at 2500g for 5 min at 4 °C to collect the supernatant. The resulting pellet was then re-extracted once. All supernatants were frozen in liquid nitrogen, freeze-dried, and stored at 80 °C for further analysis, and the sample was re-suspended with 400 lL of water. In order to detect amino acids, the samples were analyzed by high-performance liquid chromatography. 2.6. Disk diffusion test Before plating, the strains were grown in minimal medium at 37 °C with shaking to stationary phase. Then, the cultures were washed with 0.9% NaCl and concentrated for 10 times and 0.1 mL of these suspensions was placed on the top of the minimal medium plates. The analog solutions of different concentrations were applied to the disk. The plates were cultivated at 37 °C for 36 h. 2.7. Analysis L-tryptophan titer, and glucose concentration were determined as described previously (Cheng et al., 2010). Cell dry weight was determined by centrifuging the sample, washing cell pellet twice with 0.9% NaCl, and drying it at 90 °C for 24 h. Cell growth was monitored spectrophotometrically by measurement of the optical density.
Table 2. The sensitivity of 5-MT was removed by E. coli K-12-Y, suggesting that the protein YddG expressed from pSTV28-Y was activated. However, the L-tryptophan-producing strains with feedback inhibition were not highly sensitive to 5-MT. Therefore, in order to provide the successful construction of E. coli TRTH DaroP, the test PCR was necessary. 3.2. Effect of transport systems in L-tryptophan-producing strains on the accumulation of L-tryptophan The cells grown to the middle of the logarithmic phase were collected and tested in a short-term fermentation in M9 minimal medium and the intracellular tryptophan was extracted by acidic acetonitrile as described previously. The concentrations of extracellular and intracellular tryptophan were demonstrated in Fig. 2. The concentration of L-tryptophan in medium was shown in Fig. 2(a), and the recombinant strains revealed a higher concentration of extracellular tryptophan than that by strain E. coli TRTH at 6 h, and one-way ANOVA with Tukey’s test at the significance level of 0.05 proved that the results were significantly different (F > Fcrit). However, the result also indicated a higher concentration of extracellular tryptophan by E. coli TRTH DaroP than by other recombinant strains and their parent during the early phase of shortterm fermentation (<3 h). On the one hand, in the medium lacking of L-phenylalanine and L-tyrosine, the AroP permease was activated in tryptophan transport (Yanofsky et al., 1991). Therefore, in the
3. Results and discussion 3.1. Construction of recombinant plasmid and strain Many aromatic amino acid analogs, such as 5-methyltryptophan (5-MT), were transported by transport systems for aromatic amino acids, such as AroP and YddG (Kuhn, 1977; Tobias, 2010). For this reason, the bioactivity of protein YddG, expressed from recombinant plasmid pSTV28-Y, could be verified by assaying the sensitivity of 5-MT. Disk diffusion test results were presented in
Table 2 Disk diffusion test on 5-MT. Strain
E. coli K-12 E. coli K-12-Y a
Zone of inhibition (cm) of straina The concentration of 5-MT 15 lg
5 lg
3.8 <1
2.8 <1
The diameter of growth inhibition in centimeters.
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Fig. 2. Concentration of tryptophan in M9 minimal medium (a) and cytoplasma (b). The sample was collected once an hour. The extraction method of intracellular tryptophan was adopted the procedure described by Rabinowitz and Kimball (2007). The concentration of tryptophan was determined by HPLC.
M9 minimal medium, a very small amount of extracellular tryptophan could be recaptured to the cytoplasm in the case of the disruption of gene aroP. On the other hand, at the preliminary stage of cell growth, due to the limited amino acids, such as the aromatic amino acids, the participation in the biosynthesis of proteins, the biosynthesis of protein YddG and other proteins could be prevented, while E. coli TRTH-Y and E. coli TRTH DaroP-Y were required to synthesize amount of carrier protein, YddG. In this case, because of the limited biosynthesis of YddG, its ability to export L-tryptophan could be limited, as characterized by less accumulation of L-tryptophan in medium. However, as the increase in the short-term fermentation, the ability to produce aromatic amino acids by strains was improved and characterized by an increase of expression level of yddG. In this case, the bio-function of carrier protein YddG was enhanced. Therefore, more endogenetic tryptophan could be exported to the medium. Conventionally, in the fermentation process of amino acid production, some strains with altered transport systems have been used for industrial production. These strains were aimed primarily at the deregulation of the relevant biosynthetic pathway so that feedback inhibition did not work when an excess amount of a desired amino acid was intracellularly accumulated. As shown in Fig. 2(b), a lower level of endogenetic tryptophan in E. coli TRTH than in recombinant strains was observed. Krämer and his co-workers (Krämer, 1994a) have reported that three different models were developed to explain amino acid excretion: overflow metabolism, limited catabolism, and deregulated anabolism. Glutamate production in C. glutamicum was considered as a typical metabolic overflow situation. Similarly, in this study, the increasing concentration of intracellular tryptophan could be explanted as the enhanced production capacity of L-tryptophan, causing by some reasons, such as the redistribution of metabolic flow. For the strain E. coli TRTH DaroP, the amount of L-tryptophan recaptured from medium could be reduced, caused by the disruption of gene aroP. In addition, based on the fact that efflux systems, such as exporter YddG, seem to be expressed at a very low level (Doroshenko et al., 2007), L-tryptophan produced continually in the cytoplasm could not be exported to medium timely. In this case, E. coli TRTH DaroP should reveal a higher concentration of intracellular tryptophan than E. coli TRTH. Although the expression level of yddG of strains, E. coli TRTH-Y and E. coli TRTH DaroP-Y, was much higher than that of E. coli TRTH and E. coli TRTH DaroP, because of the saturation of exporter YddG, the transport capacity of YddG should probably be inactive to reduce the concentration of intracellular tryptophan. In conclusion, the modifications of transport system could result in a greater production of L-tryptophan by some reason, such
as the metabolic flux redistribution of L-tryptophan biosynthesis pathway. 3.3. Parameters of L-tryptophan-producing strains by flask-shaking fermentation Large volume of proteins and enzymes were required during the phase of cell growth and amino acid production, and the externally available amino acids, in the cytoplasm, was used directly for protein synthesis without energy consumption for anabolism (Burkovski and Krämer, 2002). In this case, the amount of the externally available amino acids was pivotal for protein synthesis, and had significant effects on biomass. The biomass of each strain by flask-shaking was demonstrated in Fig. 3. Based on the previously published report, the aromatic amino acid pools formed by AroP were the major components to participate in protein synthesis (Tobias, 2010). Therefore, the apparently lower biomass in the aroP disruption background indicated that the deficiency of aroP could be disadvantageous to cell growth due to the protein deficiency resulted from participated aromatic amino acids, as shown in Fig. 3. In the present study, the over-expression of gene yddG in L-tryptophan-producing strains was not associated with its impact on cell growth, as shown in the Fig. 3. However, over-expression of gene yddG in the wild strain showed a different result with L-tryptophan-producing strain. In the tube-fermentation, the biomass of E. coli K-12-Y (OD600 = 3.20 ± 0.06), was much lower than that of E. coli K-12 (OD600 = 3.95 ± 0.03) and E. coli K-12 carrying pSTV28 (OD600 = 3.80 ± 0.04), which indicated that over-expression of gene yddG had an adverse impact on cell growth of wild strain. The possible explanation for this phenomenon maybe the different demands of aromatic amino acids during the initial stage of cell growth. As an aromatic amino acid exporter, the over-expression of YddG resulted in a high efflux of aromatic amino acids, maintaining the normal growth of wild strains. While for the producing strains, the biosynthesis of aromatic amino acids by them was enough to satisfy the requirement of aromatic amino acids during the phase of cell growth and L-tryptophan production. In the flask-shaking fermentation, the glucose conversion rates of E. coli TRTH DaroP and E. coli TRTH-Y were 5.2% and 6.2%, respectively, and higher than their parent (4.4%), which suggested that the modification of transport system could increase the metabolic flux of L-tryptophan biosynthesis pathway. Ordinary modification characterized by over-expression of the export and inactivation of the uptake can be beneficial to the deregulating feedback inhibition during the fermentation phase, and improving the production of amino acids. However, the glucose conversion rate of E. coli
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1.8
40
1.6
35
Production of tryptophan g/l
1.4 1.2
OD660
1.0 0.8 0.6 E. coli TRTH E. coli aroP-Y E. coli TRTH-Y aroP E. coli
0.4 0.2 0.0 0
5
10
15
20
25
30
35
40
Time h Fig. 3. Cell growth curve in flask-shaking fermentation. The curve of biomass by flask shaking: the samples were diluted 1:20 with deionized water to determine the absorbance at 660 nm.
TRTH DaroP-Y was 4.6%, which did not reveal an increase as expectation, in this study. The possible reason could be due to the limited endogenetic aromatic amino acids participated in the proteins biosynthesis because this strain exported intracellular aromatic amino acids effectively and imported limited amount of extracellular amino acids to the cytoplasm. In this case, aromatic amino acids synthesized by glucose could participate in the protein synthesis rather than be exported to the medium. However, E. coli TRTH DaroP and E. coli TRTH-Y were protected from this phenomenon, which is possibly due to the fact that the concentration of aromatic amino acids participating in synthesis of proteins in E. coli TRTH DaroP and E. coli TRTH-Y cannot decrease too low to inhibit the biosynthesis of proteins. In contrast, the concentration of extracellular tryptophan in E. coli TRTH DaroP-Y in M9 minimal medium was the highest as shown in Fig. 2(a), which was different from the case in the fermentation medium. The most likely explanations for this discrepancy were shown as below. The non-growing cells in the minimal medium required less aromatic amino acids to satisfy cell growth and more to flow to the medium. At the meanwhile, in E. coli TRTH DaroP-Y, more endogenetic aromatic amino acids were exported to the medium and less extracellular aromatic amino acids were imported to the inner part of the cells These two reasons resulted in the fact that the extracellular concentration in E. coli TRTH DaroP-Y was higher when cultured in the minimal medium. In conclusion, based on the results in shaking-flask fermentation and the comparison on the glucose conversion rates of the test strains, the moderate modifications of transport system resulted in the redistribution of L-tryptophan metabolic flux, and had an effect on the cell growths.
30 25 20 15 10 5
E. coli TRTH-Y E. coli TRTH
0 -5 0
5
10
15
20
25
30
35
40
Time h Fig. 4. Production curve of tryptophan fermented by E. coli TRTH and E. coli TRTH-Y.
other hand, the result could be explained by the fact that the intrinsic transport system of E. coli has satisfied the production of L-tryptophan. Finally, in the L-tryptophan-producing strains, the disadvantage caused by increasing intracellular tryptophan concentration was not obvious. In addition, the L-tryptophan production rate, the glucose consumption rate, and biomass of strains revealed an obvious decrease at 36 h. Therefore, the fermentation was terminated at 36 h. 4. Conclusion In this study, the conclusions were proved that modifications of transport systems for L-tryptophan resulted in a greater production of L-tryptophan by the metabolic flux redistribution of L-tryptophan biosynthesis partway, and strengthen of exporter was more effective than other modifications in the fermentation of L-tryptophan, and the final output in the 30-liter fermentor was 36.3 g/L, which increased by 12.6% compared with that by E. coli TRTH. Acknowledgements This work was supported by program for Changjiang Scholars and Innovative Research Team in University (IRT1166). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012. 02.088. References
3.4. Fed-batch fermentation of E. coli TRTH-Y in 30-liter fermentor Because of relatively higher glucose conversion rate in flaskshaking fermentation, fed-batch fermentation of E. coli TRTH-Y was carried out in 30-liter fermentor to explore the production of L-tryptophan. After the fermentation for 36 h, L-tryptophan production by recombinant strain was 36.3 g/L, which increased by 12.6% compared with that by E. coli TRTH, as shown in Fig. 4. However, the result presented in the 30-liter fermentation did not exhibit a significant improvement in the production of L-tryptophan by recombinant strains. On the one hand, the composition of medium could affect the capacity of transport systems. On the
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