Tryptophan operon

Operon type

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Trp operon is an important Operon , a group of genes used or transcribed together, and also used to code and generate Tryptophan One of the components of. Tryptophan operon is present in many bacteria, but for the first time Escherichia coli Were characterized in. When there is sufficient tryptophan in the environment, it will not be used. This is an important learning gene regulation And is often used to teach the knowledge of gene regulation.

Chinese name: Tryptophan operon
Foreign name: Trp operon

Status: An important operon
Properties: a set gene

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Basic structure

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Escherichia coli Tryptophan Operon The structure is relatively simple, and it is also one of the most clearly studied operons. The structural genes are arranged as trpEDCBA in sequence, where trpGD and trpCF genes are fused. TrpE and trpG encoding O-aminobenzoic acid Synthase , trpD encoding anthranilic acid phosphoric acid Ribose Transferases , trpC encoding Indole glycerol phosphate synthase , trpF encodes isomerase, trpA and trpB code respectively Tryptophan Synthase alpha and beta Subunit 。 The upstream of trpE is Regulatory area , by Promoter 、 Manipulating gene And 162 bp Preamble sequence form. The total length of five structural genes is about 6800bp, and there is a secondary promoter at the far side of trpD Cell growth It works when excessive Trp is required.

Some G+bacteria, such as Bacillus subtilis The structure of the tryptophan operon is different. Six of the seven structural genes are arranged as trpEDCFBA, which exists in the Aromatic amino acid Hyperoperon (a ro peron), the seventh structural gene, trpG exists in folic acid In the synthetic operon, this enzyme is involved in the synthesis of Trp and folate. There are two promoters involved in regulation, one is located at the starting position of aro peron, and the other is located about 200 bp upstream of trpE^[1] 。

Regulatory action pathway

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Trp synthesis path is relatively long, consuming a lot of energy and Precursor , such as serine 、 PRPP , glutamine, etc., is one of the most expensive metabolic pathways in cells, so it is strictly regulated Tryptophan Operons play a key role. There are three main ways of regulation: Repression , weakening and the effect of end product Trp on synthetase Feedback inhibition.

Repression

Trp operon Transcription initiation The regulation of Repressor protein Implemented. The gene producing repressor protein is trpR, which is far from the trp operon gene cluster. It binds to trp to manipulate gene specific sequences and prevent transcription initiation. But repressor protein DNA The binding activity is regulated by Trp, which plays a Effector molecule The kinetic constant of Trp combination is 1~2 × 10 - 5mol · L -1 。 In the presence of high concentrations of Trp, it inhibits the formation of a Homodimer , and tightly bound to the tryptophan operon, thus preventing transcription. Repressor protein- Tryptophan The complex has a strong ability to bind to gene specific sites, and the kinetic constant is 2 × 10 - 10mol · L -1 Therefore, only 20-30 molecules of intracellular repressor proteins can play a full role. When Trp level is low, repressor protein exists in an inactive form and cannot bind to DNA. Under such conditions, the trp operon is RNA polymerase Transcription, while Trp Biosynthetic pathway Is activated.

Weakening effect

Trp operon Transcription termination The regulation of is achieved through attenuation. In E. coli trp operon, Leader region The base sequence of is composed of four fragments represented by 1, 2, 3 and 4, which can be carried out in two different ways Base pairing , 1 - 2 and 3 - 4 pairing, or 2 - 3 pairing, the 3 - 4 pairing area is exactly located Termination codon Identification area of. The leading sequence has two adjacent tryptophans Codon , when culture medium When the concentration of Trp in the medium is very low tRNA Trp is also less, so the speed of translation through two adjacent tryptophan codons will be very slow. When region 4 is transcribed, ribosome In the first region, the structure of the leading region is 2 - 3 pairing, and no 3 - 4 pairing termination structure is formed, so the transcription can continue. On the contrary, ribosomes can pass through two adjacent Tryptophan The codon, before the 4 region is transcribed, the ribosome reaches the 2 region, so that 2 - 3 can not pair, and 3 - 4 region can pair to form termination Substructure , transcription stopped.

The weakening mechanism of Bacillus subtilis has other characteristics. Because of the particularity of its tryptophan operon structure, the regulation of transcriptional initiation seems to be less important than that of transcriptional termination. Tryptophan operon expression in Bacillus subtilis is mainly activated by tryptophan RNA binding protein （Trp -activated RNA - binding Regulation of protein (TRAP). When the protein is activated to bind to tryptophan, it can bind to the upstream transcript of trpE, leading to the termination of transcription. When tryptophan concentration is low, TRAP Inactivation , transcription can continue and structural genes can be expressed. In addition, Bacillus subtilis has no load on Tryptophan TRNATrp is also very sensitive, and the latter accumulates in large quantities, which can induce the synthesis of anti TRAP protein (AT). The binding of AT with Trp activated PRAP can cancel its transcriptional termination activity. The expression of trpG is also regulated by PRAP. The activated TRAP binds to the S - D sequence overlapping with trpG, which blocks the binding of ribosomes and inhibits the transcription of trpG.

Feedback inhibition

because gene expression A certain amount of energy and precursors must be consumed, and feedback inhibition is more economical and efficient than repression and weakening. The end product Trp has a feedback inhibitory effect on the enzyme that catalyzes several steps of the branching pathway, and its 50% inhibitory concentration is respectively: o-aminobenzoate synthase, 0 0015 mmol ·L - 1 ； Phthalamic acid phosphoribosyltransferase, 0.15 mmol · L -1 ； Tryptophan synthetase ，7.7mmol·L -1 。 For normal Wild mushroom The anthranilate synthase plays a key role in the regulation of Trp synthesis, often referred to as the bottleneck enzyme; But for high yield Trp Engineering bacterium The feedback inhibition of any of the above enzymes will directly affect Trp production. Research findings Enzyme protein Mutations at some special sites can significantly reduce the sensitivity to feedback inhibition, such as serine at 38 position of anthranilate synthase is Arginine Substitution, the anti feedback inhibition ability is significantly improved when the concentration of Trp in the environment is 10 mmol · L -1 Hour enzymatic activity Under the same conditions, the activity of wild type enzyme was less than 1%. Phthalamic acid phosphoribosyltransferase 162 valine It is replaced by glutamic acid, and the anti feedback inhibition ability is also significantly improved when the environment contains 0.83 mmol · L -1 Tryptophan or 0.32 mmol · L -1 5 - Methyl- Tryptophan The enzyme activity was 3.6 times and 2.4 times higher than that of wild bacteria respectively. Chen Xiaofang and others reported a strain glutamate Seven anthranilate synthase genes of Corynebacterium Base Mutation results in 6 Amino acid residue Change, the anti feedback inhibition ability is significantly enhanced, and the Trp concentration in the environment reaches 15 mmol · L -1 The activity of o-aminobenzoate synthase hardly changed.

Genetic modification of operon

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Due to the regulation of tryptophan operon, it is impossible to have high-yield Trp strains in nature. In order to obtain high-yield Trp strains, it is necessary to Tryptophan The operon is modified to remove its regulatory effect. The early research strategy mainly relied on the traditional mutation method. After long-term efforts, some valuable research results were obtained, such as TrpR - strain, which was removed by deletion of some fragments Weakening effect , got some resistance Feedback suppression Enzymes. Many Trp producing strains were screened by random mutagenesis technology, such as Wang Jian, etc Diethyl sulfate Mutagenesis, Trp analog screening and other methods glutamate A trp strain was cultivated from corynebacterium High yield strain , shake flask fermentation for 64 h, and the yield of trp reached 7.28 g · L -1 。

Although the traditional mutation method is effective, its shortcomings are obvious, such as heavy workload, low efficiency, Mutant Growth of bacteria, environmental Tolerance And the genetic stability is worse than that of wild type strains. genetic engineering The establishment and development of technology provide a new technical platform for the transformation of tryptophan operons. Adopted by Tribe et al. in 1979 DNA recombination technology yes Escherichia coli Transformed, amplified trp operon, fermented for 12 h, and produced 1 g · L acid -1 Although the acid production is not very high, its significance is very significant, which created the genetic engineering technology in Trp biosynthesis First application. Later, Aiba, etc. will have Tryptophan Operon plasmid was introduced into Escherichia coli, fermented for 27 h, and supplemented O-aminobenzoic acid And get trp 6.2 g · L -1 。 Ikeda et al. amplified branching pathways by constructing stable plasmids Rate limiting enzyme And transform the center Metabolic pathway And Trp production reaches 58 mg · L -1 Strains. In addition to amplification expression Operon gene The rational design and transformation of it has also begun to attract attention. Mutations at certain specific sites of known enzyme molecules can lead to Feedback inhibition susceptibility Therefore, we can consider the use of genetic engineering technology for tryptophan operon Structural gene Rationally modify it to reduce its sensitivity to feedback inhibition, but there is still a lack of successful examples, mainly because the existing enzyme molecular feedback inhibition structure and Functional relationship The data is insufficient to meet the needs.

Metabolic Engineering Theory

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In 1991, Bailey used metabolic engineering Describe utilization DNA recombination technology Cellular Enzyme reaction , material transportation and regulation function Genetic manipulation To improve cells biological activity The process of metabolic engineering marks a turning point in the development of metabolic engineering into a systematic discipline. Metabolic engineering, also known as pathway engineering, is different from traditional single gene expression (the first generation genetic engineering ）And gene directed mutation（ Second generation genetic engineering ）, is to target cells biochemical reaction Of Metabolic network The technology of decoration Polygene Design and modify the inherent metabolic pathway and Genetic trait And endow cells with more superior and even new product production quality. Metabolic engineering is improving host cell original metabolite Production New substances Expansion and construction of new metabolic pathways and production Metabolites as amino acid 、 antibiotic , vitamins and degradation Environmental pollutants And many other aspects show broad application prospects. Theoretically, improving Trp production rate is the primary task of metabolic engineering, which requires a good understanding of Trp biosynthesis and the dissimilation pathway of intracellular control of Trp metabolism, as well as a broader Microbial metabolism The effective mathematical model 。 Early models mainly considered some aspects of tryptophan operon dynamics, and only a few research model The three mechanisms of tryptophan operon are comprehensively considered. Xiuzhilong Et al. introduced the theory of metabolic engineering into the field of trp metabolic analysis and established a suitable mathematical model. It was found that the level of repression and the intensity of enzyme feedback inhibition seriously affected the target variable, namely trp concentration, under the condition of stable metabolism. The dynamic model proposed by Santillan et al. uses the Second Lyapunov's method analysis, and compares and verifies the performance of wild strains and several improved strains (the feedback inhibition and weakening effect of anthranilate synthase are respectively relieved), and concludes that the feedback inhibition of enzyme system stability It plays an important role, but the weakening effect has little effect, mainly when Trp nutrition changes. These two models have certain Representativeness They take into account the feedback inhibition of enzymes, which has certain guiding significance for Trp biosynthesis; However, its shortcomings are also obvious. Only the feedback inhibition of o-aminobenzoate synthase is considered, and other enzymes are not considered. Another disadvantage is the lack of high yield Tryptophan Strains to verify^[2] 。

reference data

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Transcription (Bacteria Eukaryote )
Transcriptional regulation	Prokaryote Operon （ Lactose operon 、 Tryptophan operon 、 Galactose operon ） · Repressor （ Lactose repressor 、 Tryptophan repressor ） Eukaryote Histone Modifying Enzymes （ Histone / Nucleosome ）： histone methylation / Histone methyltransferase （ EZH2 ） · Histone demethylase · Histone acetylation and deacetylation （ Histone deacetylase HDAC1 · Histone acetyltransferase ） DNA methylation （ DNA Methyltransferase ） Chromatin remodeling ： CHD7 Both Transcription co regulators （ Excipient 、 Auxiliary repressor ） · Inducer
start-up	Promoter （ pribnow box 、 TATA box 、 BRE 、 CAAT box 、 Answering element ） · Enhancer （ E-box 、 Response element ）·Insulators· Silent Son
Start (bacteria Eukaryote ）	Transcription start site
extend	Bacteria RNA polymerase ： rpoB Eukaryote RNA polymerase ： RNA Polymerase II
Termination (bacteria Eukaryote ）	Terminator · Internal termination · ρ factor
See also: Transcriptional and Posttranscriptional Modification Diseases Biochemical: Protein Biosynthesis ： Gene expression regulation （ Cell cycle proteins ， Oncogene ） · Transcription (Nucleic acid, RNA ， Transcription Factors and Intracellular Receptors ， Repeating sequence ， Post transcriptional modification ）·Translation（ Ribosomal subunit ， Ribonucleoprotein ） · Ubiquitin cross-linking enzyme · Sorting protein · Molecular chaperone ·Structure（ protein domain ， 1° ， 2° ， 2° ， 3° ， 4° ）

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