| Essential amino acid tryptophan is an important nutritional component in human and animal health. It contains three types of isomer:L-type, D-type, DL-type. L-tryptophan is one of8essential amino acids which cannot be synthase by human and animal body. Hence, L-tryptophan can only be absorbed from green plants and/or by gut bacteria. It has been well established that the all tryptophan in the body of human and animals is absorbed only from the food. Since D-tryptophan cannot be utilized by human and animal body, the current work mainly focused on the improvement of L-tryptophan production. With the rapid progress in the human and animal nutrition regarding the tryptophan metabolism, its application is also gradually extending from food and feed additives to medicine.Based on aromatic amino acid metabolism pathway of Escherichia coli, this study introduced the Red recombinant system to knockout trpR genes in the genomes of E. coli in order to remove the tryptophan synthesis feedback repression, which is the restricted step in the pathway of tryptophan synthesis network. On the other hand, we rebuild a plasmid with higher the copy numbers in E. coli which contains the expression of tryptophan operon gene to improve the fermentation level of tryptophan. In this thesis, the main results are described as follows:(1).Construction of trpR gene mutations using Red recombinant systemThe key step of tryptophan synthesis in E. coli is behind of the center step in the metabolic pathway, including a common pathway and a branched pathway. Enzymes used in branched pathway are mainly controlled by the tryptophan operon. TrpE and trpD are genes coding anthranilic acid synthase (AS), which largely controlled by feedback inhibition. Anthranilic acid synthase is one of the key enzymes in the tryptophan synthesis process in E. coli. Its activity is controlled by trpR. Once trpR expressed and combined with tryptophan, it can repress the transcriptional process in the tryptophan operon. Therefore, if we knockout trpR genes in the genomes of E. coli, the feedback inhibition in the process of tryptophan synthesis could be released and in subsequences to achieve the purpose of increasing production of tryptophan.(2) Reconstruction of plasmid mc33Current plasmid has tryptophan operon trpABCDE series arranged and expressed in one plasmid. In current study, we constructed two recombinant plasmids, pSU18-trp operon and pUC19-serA/aroG The former contains trpABCDE and the later contains the intermittent sera and aroG After transforming the new plasmids into E. coli, production level of tryptophan in shake flask reached0.1mg/L after15h fermentation.(3) Establishing HPLC method for the determination of L-tryptophan in fermented liquid.Traditional method for determination of L-tryptophan included paper chromatography, thin layer chromatography, colorimetric method and high performance liquid chromatography (HPLC) method and amino acids analysis method. The most commonly used methods in laboratory research are diaminobenzene formaldehyde method and derivatives HPLC method. However, the two methods showed their limitations, mainly due to the largely utilization of the sulfuric acid as chromogenic agent. It can be dangerous in the laboratory. Therefore, HPLC method has been gradually adopted as the main measurement method for the determination of L-tryptophan. According to the specific light absorption of L-tryptophan in UV at278nm, determination of tryptophan can be conducted on basis of the use of photoelectric diode array detector (DAD) to detect absorption peak area under the wavelength. This study developed a HPLC operating conditions:the SunFireTMC18column (5microns,150mm x4.6mm) was used as the separation column; mobile phase proportion is0.03%KH2PO4solution:methanol (V:V)=90:10; flow rate was1mL/min and the detection wavelength was at278nm. Column temperature was39℃and the sample injection was20ul. (4) Whole sequencing and analysis of the original plasmid MC33and the original E. coli strain genome. Due to the confidential agreement, those part of results were not fully included in current thesis... |
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