The tryptophan manipulator

Tryptophan manipulator

The structure of the E. coli tryptophan manipulator is relatively simple, and it is also the most well-studied manipulator, with the structural genes in order of trpE, trpD, trpC, trpB, and trpA, in which trpG is fused with trpD and trpC is fused with trpF. trpE and trpG encode phthyllo-amino-benzoate synthase, trpD encodes phosphoribosyl transferase, trpC encodes indolylglycerol phosphate synthase, and trpF encodes isomerization enzyme. trpE and trpG encode o-aminobenzoate synthase, trpD encodes o-aminobenzoate phosphoribosyltransferase, trpC encodes indole glycerophosphate synthase, trpF encodes isomerase, and trpA and trpB encode the α- and β-subunits of tryptophan synthase, respectively. trpE has a regulatory region upstream of trpE, which consists of the promoter, the manipulator gene, and 162 bp of the leading sequence. the five structural genes have a total length of approximately 6800 bp, and there is a secondary promoter distal to trpD. There is also a secondary promoter that functions when excess Trp is required for cell growth.

Some G+ bacteria, such as Bacillus subtilis tryptophan manipulator, have different structures. 6 of the 7 structural genes, trpE, trpD, trpC, trpF, trpB, and trpA, are in the aromatic amino acid super-operon containing 12 structural genes, and the 7th structural gene, trpG, is in the folate synthesis manipulator. The seventh structural gene, trpG, is found in the folate synthesis manipulator, which is involved in the synthesis of Trp and folate. Two promoters are involved in its regulation, one at the start of the aro operon and the other about 200 bp upstream of trpE. The Trp synthesis pathway is long and consumes large amounts of energy and precursors, such as serine, PRPP, and glutamine, and is one of the most expensive metabolic pathways in the cell, and is therefore tightly regulated, with the tryptophan operon playing a key role. There are three main modes of regulatory action: deterrence, weakening, and feedback inhibition of synthase by the end product Trp.

Deterrence

The regulation of the transcription initiation of the trp manipulator is achieved by deterrent proteins. The gene that produces the deterrent protein is trpR, which is distant from the trp operon gene cluster. It binds to trp manipon gene-specific sequences and prevents transcription initiation. However, the DNA-binding activity of trp is regulated by Trp, which acts as an effector molecule and binds with a kinetic constant of 1-2 × 10- 5mol-L-1. In the presence of high concentrations of Trp, the trp-tryptophan complex forms a homodimer and binds tightly to the tryptophan operon, thus preventing transcription. The ability of the Trp-tryptophan complex to bind to gene-specific sites is very strong, with a kinetic constant of 2 × 10- 10mol-L - 1, so that only 20-30 molecules of Trp in the cell can be fully functional. When Trp levels are low, the repressor protein exists in an inactive form and is unable to bind DNA. under these conditions, the trp manipulator is transcribed by RNA polymerase, and the Trp biosynthetic pathway is activated.

Attenuation

The regulation of transcription termination of the trp manipulator is achieved by attenuation. In the E. coli trp operon, the base sequence of the leading region consists of four segments denoted 1, 2, 3, and 4 that are capable of base pairing in two different ways, 1 - 2 and 3 - 4 pairing, or 2 - 3 pairing, with the 3 - 4 pairing region being located right in the recognition region of the termination codon. The leading sequence has two adjacent tryptophan codons, when the concentration of Trp in the medium is very low, the tRNATrp loaded with Trp is also less, so that the translation through the two adjacent tryptophan codons will be very slow, when the 4 region is transcription is completed, the ribosome stays in the 1 region, the structure of the leading region at this time is 2 - 3-pairing, do not form a termination structure of the 3 - 4-pairing, so that the transcription can continue to proceed. On the contrary, the ribosome can pass through two neighboring tryptophan codons smoothly, and before the 4 region is transcribed, the ribosome reaches the 2 region, so that the 2 - 3 can not be paired, and the 3 - 4 region can be paired to form a terminator structure, and the transcription stops.

The mechanism of weak action of B. subtilis is otherwise characterized. Because of the specificity of its tryptophan manipulator structure, the regulation of transcription initiation seems to be less important than the regulation of transcription termination. The expression of the B. subtilis tryptophan manipulator is mainly regulated by the tryptophan-activated RNA-binding protein (TRAP). This protein is activated by binding to tryptophan and can bind to trpE upstream transcription products, leading to transcription termination. When tryptophan concentration is low, TRAP is inactivated and transcription can continue and structural genes are expressed. In addition, B. subtilis is sensitive to unloaded tryptophan tRNATrp, which accumulates in large quantities and induces the synthesis of anti-TRAP proteins (anti-TRAP,AT). AT binds to Trp-activated TRAP and abolishes its transcription termination activity. trpG expression is also regulated by TRAP, and the activated TRAP binds to S-D sequences that overlap with those of trpG and impede the binding of ribosomes, inhibiting trpG expression. ribosome binding and inhibits trpG transcription.

Feedback inhibition

Because gene expression inevitably consumes a certain amount of energy and precursors, feedback inhibition is more cost-effective and efficient than deterrence and weakening. The 50% inhibitory concentrations of the end product Trp on the enzymes catalyzing several steps of the branching pathway were: o-aminobenzoate synthase, 0.0015 mmol-L-1; o-aminobenzoate phosphoribosyltransferase, 0.15 mmol-L-1; and tryptophan synthetase, 7.7 mmol-L-1. For the common wild strain, o-aminobenzoate synthase played a key role in regulating Trp synthesis, and it was also a key regulator of Trp synthesis. For wild strains, o-aminobenzoate synthase plays a key role in the regulation of Trp synthesis and is often referred to as the bottleneck enzyme; however, for high Trp-producing engineering bacteria, the feedback inhibition of any of the above enzymes will directly affect the Trp yield. It was found that mutation of some special sites of enzyme proteins could lead to a significant decrease in the sensitivity to feedback inhibition, such as o-aminobenzoic acid synthase, the serine at position 38 was replaced by arginine, which significantly increased the ability to resist feedback inhibition, and the enzyme activity was not affected when the Trp concentration in the environment was 10 mmol-L-1, while the activity of the wild-type enzyme was less than 1% under the same conditions. When valine at position 162 of o-aminobenzoic acid phosphoribosyltransferase was replaced by glutamic acid, the anti- feedback inhibition ability was also significantly improved, and the enzyme activity was 3. 6 times and 2. 4 times that of the wild-type bacterium, respectively, when the environment contained 0. 83 mmol-L - 1 tryptophan or 0. 32 mmol-L - 1 5 - methyl- tryptophan. Chen Xiaofang et al. reported that a strain of Corynebacterium glutamicum o-aminobenzoate synthase gene with 7-base mutation resulted in 6 amino acid residue changes, and the resistance to feedback inhibition was significantly enhanced, and the activity of o-aminobenzoate synthase was almost unchanged when the Trp concentration in the environment reached 15 mmol-L-1. Due to the regulatory role of the tryptophan manipulator, it is impossible to exist high-yielding Trp strains in nature, and in order to obtain high-yielding Trp strains, it is necessary to modify the tryptophan manipulator to deregulate its regulatory role. Early research strategies mainly relied on traditional mutagenesis methods, and after a long period of effort, some valuable research results were obtained, such as the acquisition of TrpR - strains, the disarming of the weakening effect by deletion of certain fragments, and the acquisition of some enzymes resistant to feedback inhibition. Many Trp-producing strains were screened by random mutagenesis, such as Wang Jian et al. who developed a high trp-producing strain from Corynebacterium glutamicum by diethyl sulfate mutagenesis, Trp analog screening, etc., which produced 7.28 g-L-1 of trp after shaking-flask fermentation for 64 h.

Traditional mutagenesis, although effective, has some obvious drawbacks, such as The workload is large, the efficiency is low, and the bacterial growth, environmental tolerance and genetic stability of mutant strains are poorer than those of wild-type strains. The establishment and development of genetic engineering technology provides a new technical platform for the transformation of tryptophan manipulator. 1979 Tribe et al. used DNA recombination technology to transform E. coli, amplified trp manipulator, fermented for 12 h, and produced 1 g-L - 1 acid, although the amount of acid production is not very high, but its significance is very significant, thus creating a genetic engineering technology in the application of Trp biosynthesis. The application of genetic engineering technology in Trp biosynthesis was thus pioneered. Subsequently, Aiba et al. introduced a plasmid with a tryptophan manipulator into Escherichia coli, fermented for 27 h, and supplemented it with o-aminobenzoic acid to obtain trp 6.2 g-L-1. Ikeda et al. constructed a stabilized plasmid, amplified the branching pathway rate-limiting enzyme, and modified the central metabolic pathway, and obtained a strain that produced 58 mg-L-1 of trp. In addition to amplification and expression of manipulator genes, their rational design and modification have also attracted attention. It is known that mutations in certain specific sites of enzyme molecules can lead to a decrease in sensitivity to feedback inhibition, so genetic engineering techniques can be considered to rationally modify the tryptophan manipulator structural genes to reduce their sensitivity to feedback inhibition, but Z so far lack of successful examples, mainly because of the insufficient information on the relationship between the feedback inhibitory structure and function of the existing enzyme molecules can not meet the needs. In 1991, Bailey used metabolic engineering to describe the process of using DNA recombination technology to genetically manipulate the enzymatic reactions, material transport, and regulatory functions of cells, thereby improving cellular bioactivity, marking a turning point in the development of metabolic engineering into a systematic discipline. Metabolic engineering, also known as pathway engineering, distinguishes itself from traditional single-gene expression (first-generation genetic engineering) and gene-directed mutagenesis (second-generation genetic engineering), and is a technology that purposely modifies the metabolic network of the cell's biochemical reactions, designing and modifying the metabolic pathways and genetic traits inherent to the cell at a polygenic level and endowing the cell with superior or even brand-new product production qualities. Metabolic engineering has shown great promise in increasing the yield of existing metabolites in host cells, generating new substances, expanding and constructing new metabolic pathways, producing metabolites such as amino acids, antibiotics, vitamins, and degrading environmental pollutants. Theoretically improving Trp yield is the first task of metabolic engineering, which requires a good understanding of Trp biosynthesis and of the heterogeneous pathways controlling Trp metabolism within the cell, as well as an effective mathematical model describing these pathways within a broader microbial metabolic network. Early models mainly considered one aspect of tryptophan manipulator dynamics, and there are only a few research models that combine the three mechanisms of action of tryptophan manipulators. Xiu et al. introduced the theory of metabolic engineering into the field of trp metabolism analysis and established an appropriate mathematical model, finding that the level of deterrence and the strength of feedback inhibition of the enzyme severely affect the target variable, i.e., the trp concentration, under metabolic stability.The kinetic model proposed by Santillan et al. was analyzed using Second Lyapunov's method analysis, by comparing and validating the performance of wild and several modified strains (o-aminobenzoic acid synthetase feedback inhibition and weakening effect were lifted respectively), and concluded that the feedback inhibition of the enzyme plays an important role for system stability, while the weakening effect has a smaller impact and mainly acts when Trp nutrition is changed. These two models are representative, and they consider the feedback inhibition of enzymes, which is of some significance for Trp biosynthesis; however, their shortcomings are also obvious, only the feedback inhibition of o-aminobenzoic acid synthetase was considered, and other enzymes were not taken into account, and another shortcoming is that there is a lack of high tryptophan-producing strains to be verified.