One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli
© Gu et al; licensee BioMed Central Ltd. 2012
Received: 19 December 2011
Accepted: 2 March 2012
Published: 2 March 2012
L-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.
The 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.
The one step inactivating and promoter swapping is an efficient method for metabolic engineering. This method can also be applied in other bacteria.
L-tryptophan is an essential aromatic amino acid for humans and animals which can be used as food additive, infusion liquids, pellagra treatment, sleep induction and nutritional therapy [1, 2]. Since the chemical synthesis of L-tryptophan has many disadvantages such as nonrenewable toxic raw materials and racemic mixtures of products, microbial fermentation of L-tryptophan has become attractive alternative. E. coli, a widely used production host that possesses clear genetic background, convenient metabolic engineering tools and fast growth in cheap media, has attracted many attentions for the production of L-tryptophan and other aromatic compounds [3–7].
Otherwise, the transcription and expression of tryptophan operon is pivotal to obtain high L-tryptophan accumulation as well . Promoter swapping allowed researchers to replace a wild type promoter with the one that has been designed for a increased or controlled transcription strength while retaining the natural genetic context of a gene or an operon in the genome . Consequently, by promoter swapping and engineering, the targeted metabolites can be elevated. For example, to maximize the threonine production, Lee at al. created an L-threonine producing strain by replaced three different chromosomal promoters. After replaced the native promoter of the ppc gene with trc promoter in the chromosome, the engineered strain showed a higher PPC flux than the wild type, and therefore resulting 27.7% increased threonine production . In another study, Alper et al. found a correlation between promoter strength and lycopene production. By introducing a promoter library that was created by error-prone PCR into E. coli to replace native promoter of phosphoenolpyruvate carboxylase or deoxy-xylulose-phosphate synthase, they identified a suitable promoter for lycopene production .
In this study, we first constructed a basic L-tryptophan-synthetic strain by inactivation of the trpR, tnaA and ptsG, expressing in plasmids the feedback resistant aroG, trpE (aroG FR and trpE FR respectively), and tktA genes in wild E. coli K-12 W3110. Then, we inactivated the tryptophan attenuator and replacing the original trp promoter of tryptophan operon with a novel promoter cluster consisted of five core-tac-promoters aligned in tandem (5CPtacs promoter cluster) in one step. The resulting strain GPT1002 showed higher transcription of tryptophan operon genes and more L-tryptophan accumulation than the parent strain.
Results and discussion
Construction of the basic L-tryptophan-synthetic E. Coli GPT1001
Development of L-tryptophan producing E. coli strains
L-tryptophan (mg l-1)
0.12 ± 0.01
0.14 ± 0.02
W3110 (∆trpR::FRT)/pCL1920-trpE FR
64.46 ± 2.17
W3110 (∆trpR::FRT)/pCL1920-trpE FR -aroG FR
736.83 ± 3.98
1018.98 ± 1.89
W3110 (∆trpR::FRT, ∆tnaA::FRT)/pTAT
1188.20 ± 2.56
W3110 (∆trpR::FRT, ∆tnaA::FRT, ∆ptsG::FRT)/pTATa
1208.82 ± 1.33
Alleviating the feedback repression of the product increased the expression of the key enzymes in the tryptophan biosynthesis pathway, while provision of more precursors would enable the enhanced metabolic flux. tktA gene, encoding a transketolase in pentose phosphate pathway, and overexpression of this gene in E. coli was proved to supply more E4P, a precursor of L-tryptophan . Otherwise, carbon flux distribution analysis at the node in wild E. coli indicated that phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is the largest consumer of PEP, while the relative carbon flux directed to aromatic amino acid biosynthesis is only around 1.5% of the PTS consumed . Therefore we knocked out ptsG, which encodes the IIBC component of glucose-specific PTS system, to provide more PEP. In our base strain, we performed modification of the host to increase the levels of precursors PEP and E4P, while PRPP and Lserine are also building blocks for L-tryptophan. Therefore, increasing the availability of L-serine by amplification of the deregulation serA gene  and PRPP by overexpresssion of prs and ywlF genes involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate  should be useful for high L-tryptophan accumulation. Finally, we knocked out the gene tnaA, which encodes a tryptophanase that catalyzes the reaction of L-tryptophan back into indole . The resulting L-tryptophan-synthetic strain was named GPT100. Then we transformed plasmid pTAT into E. coli GPT100 and constructed strain GPT1001. This strain was able to produce 1.3 g l-1 L-tryptophan in batch cultivation and was therefore used as base strain for further experiment.
One-step L-tryptophan attenuator inactivation and promoter swapping
The expression of tryptophan biosynthesis operon was negatively regulated by the attenuator downstream of the promoter operator site until tryptophan starvation is severe. However, simply removal of the attenuator probably cannot reach a sufficient expression of the tryptophan operon genes . Therefore it is essential to improve the expression of genes in tryptophan operon at the same time of inactivating the attenuator.
Characterization of tryptophan operon transcription in E. Coli GPT1002
In addition, the transcription level of the aroG gene in the strain GPT1002 was also significantly upregulated by 9.29 ± 0.32 fold. In order to determine whether the different expression levels of trpE and aroG on the plasmid pTAT lead to this phenomenon, we analyzed the expression of trpE, aroG and trpD in the strain GPT101 and GPT100, the parent strains of GPT1002 and GPT1001 without the recombinant plasmid pTAT, respectively (Figure 3B). The relative transcription of three genes in both GPT101 and GPT100 were similar to the strain harboring pTAT, and therefore excluded the impact of plasmid pTAT. Since AroG protein is critical of controlling the carbon flow into aromatic amino acid biosynthesis pathway [28, 29], more experiments such as metabolic flux analysis should be helpful to find out the reason of high aroG transcription.
Production of L-tryptophan by E. Coli GPT1002
Through promoter swapping, a wild type promoter could be replaced with the one that has been designed for increased or controlled transcription strength [31, 32]. However, a normal strong promoter is sometimes not sufficient for downstream gene expression. Recently, we developed a promoter cluster, by which the core tac promoter region was arranged repetitively in tandem. This method can improve the expression of desired genes without increasing the copy number of the gene. When the repetition number was 5, the transcription strength increased almost 4 fold . In this study, the 5CPtacs promoter cluster was swapped into the upstream region of tryptophan operon, which resulted in an ideal result. Besides E.coli, this promoter swapping method can also be applied in many other bacteria and other regions of the chromosome. Nevertheless, the choice of swapping region in the chromosome is very important. When the swapping region contains indispensable gene or essential regulation elements, the swapping should be careful.
We developed a method for one step inactivating the tryptophan attenuator and promoter swapping. The engineered E. coli GPT1002 showed strong transcription capability and L-tryptophan accumulation. The L-tryptophan production of GPT1002 can be further improved through strain improvement and fermentation process optimization. The one step gene inactivating and promoter swapping is an efficient method for metabolic engineering and can also be applied in other bacteria.
Bacterial strains and plasmids construction
Strains and plasmids used in this study
F - , λ - , rph-1, IN (rrnD, rrnE)
F - , endA1, hsdR17 (r K - , m K + ), supE44, thi-l, λ - , recA1, gyrA96, ΔlacU169 (Φ80lacZ ΔM15)
W3110 ΔtrpR::FRT, ΔtnaA::FRT, ΔptsG::FRT
GPT100 with tryptophan attenuator deletion and trp promoter swapping by 5CPtacs promoter cluster
GPT100 containing pTAT
GPT101 containing pTAT
pCL1920 containing aroG FR , trpE FR , and tktA
bla, helper plasmid
bla and cat, helper plasmid
pCL1920 containing 5CPtacs promoter cluster and gfp
pBluescript SK-, containing kan and 5CPtacs promoter cluster
Primers used in this study
Primers for RT-PCR
Plasmid pBluescript SK- was served for constructing recombinant vector pKMT. The kan gene and the 5CPtacs promoter cluster were obtained with the kan-F and kan-R, and Mtac-F and Mtac-R as the primers and the plasmids pKD4 and p5TG as the templates separately using the TransTaq DNA Polymerase High Fidelity from TransGene Biotech (Beijing, China). Next, the PCR products were digested with Bam HI/Eco RI and Eco RI/Sac I respectively, and then ligated into the vector pBluscript SK- and constructed the plasmid pKMT.
Three genes trpR, tnaA, and ptsG, which encoded trp operon repressor, tryptophanase, and glucose-specific PTS enzyme IIBC components respectively, were inactivated in turn using the one-step inactivation method . Primers trpR-F and trp-R, tnaA-F and tnaA-R, and ptsG-F and ptsG-R, template plasmids pKD3 for ptsG and pKD4 for trpR and tnaA were used to obtain the linearized DNA flanked by FLP recognition target sites and homologous sequences for genes deletion. The PCR was performed in an automated thermocycler (Bio-Rad, Hercules, CA, USA), and then PCR products were gel-purified and digested with Dpn I. Electroporation was done according to the manufacturer's instructions by using 25 ml of cells and 10-100 ng of PCR product to transform resistance gene cassette into the cells expressing the Red recombinase before. Shocked cells were added to 1 ml SOC cultures, incubated 1 h at 37°C, and one-half was spread onto agar to select chlorampenicol resistant or kanamycin resistant transformants. Positive clones on the plates were verified by PCR using the primers trpRtest-F and trpRtest-R, tnaAtest-F and tnaAtest-R, and ptsGtest-F and ptsGtest-R separately. The chlorampenicol or kanamycin cassette was removed with the helper plasmid pCP20. The final strain E. coli K-12 W3110 with three mutations (ΔtrpR ΔtnaA ΔptsG) was named GPT100.
One-step of L-tryptophan attenuator inactivation and promoter swapping
The DNA fragment for next promoter replacement was amplified using plasmid pKMT as the template with the primers Trp-F and Trp-R and the fragment containing kan gene and 5CPtacs promoter cluster was transformed into GPT100 by electroporation, incubated for 1 hours at 37°C, and spread onto agar to select kanamycin resistant transformants. The strategy of plasmid pKMT construction and promoter swapping were listed in Figure 2. The positive clones were verified by PCR using the primers trptest-F and trptest-R, and named GPT101. Then we transformed the plasmid pTAT into GPT101 and the GPT100 respectively, and resulting to the recombinant strain GPT1002 and control strain GPT1001 for next experiments.
Quantitative real-time reverse transcription (RT)-PCR analysis
Samples for mRNA preparation were cultivated 6 h after the addition of 0.1 mM IPTG if necessary. Total cellular RNA was extracted by the RNA simple Total RNA Kit (TIANGEN, Beijing, China) as described by the manufacturer. The quantity and purity of RNA were determined by spectrophotometrically at A260 and A280. The reverse transcription was performed using primers Random 6 mers and Oligo dT by the PrimeScript RT reagent Kit (TaKaRa, China) according to the manufacturer. RT- PCR was performed with SYBR Premix Ex TaqII (TaKaRa, China) followed the protocol of the Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). The RT-PCR measurement was repeated three times for each sample. The trpE, trpD, trpC, trpB, trpA, aroG genes transcripts primers were listed in Table 3 and gapA encoding D-glyceraldehyde-3-phosphate dehydrogenase transcript selected as internal standard was amplified with gapART-F and gapART-R.
Strains for cloning and inoculums were grown in Luria-Bertani media (1% tryptone, 0.5% yeast extract and 1% NaCl) at 37°C for 8-12 h supplemented with the appropriate antibiotic (ampicillin (100 mg l-1), chloramphenicol (17 mg l-1), kanamycin (25 mg l-1), spectinomycin (50 mg l-1)) when necessary. For fermentation, the seed medium contained (per liter) glucose (20 g), MgSO4·7H2O (5 g), KH2PO4 (1.5 g), (NH4)2SO4 (10 g), yeast extract (15 g), FeSO4·7H2O (15 mg), sodium citrate dehydrate (0.5 g), Vitamin B1 (100 mg). The fermentative medium contained (per liter) glucose (20 g), MgSO4·7H2O (5 g), KH2PO4 (2 g), (NH4)2SO4 (4 g), yeast extract (1 g), FeSO4·7H2O (100 mg), sodium citrate dehydrate (2 g). A single clone was pre-cultured in 5 ml Luria-Bertani medium at 37°C and on a rotary shaker at 200 rpm overnight. 1 ml overnight cells were inoculated into 50 ml seed medium and cultured for 8-12 hours, and then 10% (v/v) seed cultures for batch cultivation were incubated into 50 mL fermentation medium at 37°C with the initial glucose concentration 20 g l-1. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at the final concentration of 0.2 mM. For fed-batch fermentation, a stirred 5-l glass vessel with the BioFlo310 modular fermentor system (New Brunswick Scientific, Edison, NJ, USA) was used. The inoculum ratio was 10% (v/v). When glucose concentration in the medium was below 10 g l-1, feeding solution containing 500 g l-1 glucose was supplied to the medium. The culture temperature was 37°C, and the pH was controlled at 6.8 with NH3·H2O. The dissolved oxygen concentration was kept at 30% via changing fermentor agitation speed and aeration rate.
Cell growth was monitored by OD600 with a spectrophotometer (Shimazu, Japan). Glucose was quantitatively analyzed by high-performance liquid chromatography (HPLC; Shimazu, Japan) equipped with a column of Aminex HPX-87H Ion Exclusion particles (300 mm × 7.8 mm, Bio-Rad, Hercules, CA, USA). Samples were centrifuged at 12 000 rpm for 5 min and then filtrated with a 0.22 μm aqueous membrane. The mobile phase was 5 mM sulfuric acid (in Milli-Q water) with the flow of 0.6 ml min-1 and the column was maintained at 65°C. L-tryptophan was determined by the method of fluorometric determination .
This work was financially supported by a grant from the National Natural Science Foundation of China (31070092) and a grant of the National Basic Research Program of China (2011CB707405).
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