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L-tyrosine (Tyr) is an important nutritional essential amino acid, which plays an important role in the metabolism, growth and development of human and animals, and is widely used in food, feed, medicine and chemical industries. It is often used as a nutritional supplement for patients with phenylketonuria, as well as a raw material for the preparation of polypeptide hormones, antibiotics, L-dopa, melanin, p-hydroxycinnamic acid, p-hydroxystyrene and other pharmaceutical and chemical products. However, with the discovery of more high-value-added L-tyrosine derivatives such as danshensu, resveratrol, hydroxytyrosol, etc. in organisms, L-tyrosine is increasingly developing towards platform compounds.
1. L-tyrosine is used in medicine to treat hyperthyroidism.
2. L-tyrosine is an important biochemical reagent and the main raw material for the synthesis of peptide hormones, antibiotics, L-dopa and other drugs.
3. L-tyrosine can be used as food additive.
The early production of L-tyrosine mainly depends on protein hydrolysis. However, the protein hydrolysis method has been eliminated due to its shortcomings such as limited material sources, complex process and products, and long cycle. L-tyrosine is mainly produced by enzyme method, microbial fermentation method, extraction method and chemical method.
1. Enzymatic method
Enzymatic method, also known as microbial transformation method, is mainly to convert phenol, pyruvate and ammonia or phenol, L-serine into L-tyrosine by using tyrosine phenol-lyase (TPL, EC 4.1.99.2) in microbial cells. The TPL with high enzyme activity that has been studied extensively mainly comes from the microorganism Erwinia herbicola, Citrobacter intermedium, Citrobacter freundii and Thermophilic bacteria. Lee and Hsiao of Genex Company first used the serine hydroxymethyltransferase of Klebsiella aerogenes and the tyrosine phenol lyase of Erwinia herbicola ATCC 21434 to couple the reaction of synthesizing L-serine with glycine as the substrate and the reaction of synthesizing L-tyrosine with L-serine as the substrate in 1986. Add 0.32% phenol, 0.25 M glycine, 0.5 mM pyridoxal 5-phosphate, 0.056 M in 500 mL reaction system β- Mercaptoethanol, 1.7 mM tetrahydrofolate. When the reaction was started with 37% formaldehyde at pH 7.0 and 37 ℃, L-tyrosine 26.3 g/L was produced after 16 hours, and the conversion rate of glycine reached 61.4%. However, the stability of this process is poor and glycine has a strong inhibitory effect on TPL activity. Considering the disadvantages of poor enzyme activity and stability in the reaction process, the use of DNA shuffling technology to improve the stability of TPL has also attracted attention in recent years. Eugene et al. of KRIBB in South Korea obtained AS6 mutant with catalytic activity increased by three times and half-inactivation temperature increased by 11.2 ℃ through random mutation screening and staggered DNA shuffling of TPL in Symbiobacterium Toebii. The sequencing results showed that there were T129I or T451A mutations in the catalytic active region, and three mutations including A13V, E83K and T407A were greatly helpful to improve the thermal stability. Kim et al ColiBL21 (DE3) overexpressed TPL with improved catalytic activity and thermal stability, and prepared the catalytic crude extract. In a 2.5 L flow plus reactor system, 2.2 M phenol, 2.4 M sodium pyruvate, 0.4 mM pyridoxal 5-phosphate and 4 M ammonium chloride were added in batches and filled with nitrogen above the reaction tank to reduce the oxidation of the substrate. After 30 hours of reaction at 40 ℃, 130 g/L L-tyrosine could be accumulated, and the conversion rate of phenol could reach 94%.
2. Microbial fermentation method
The microbial fermentation method usually uses glycerol, glucose and other biomass carbon sources as raw materials, and accumulates L-tyrosine through the fermentation of excellent microbial strains under appropriate conditions. Early studies often used artificial mutation to select L-tyrosine-producing strains, such as screening L-phenylalanine or L-tryptophan deficiency or anti-feedback inhibition strains. However, the ability of most microorganisms to accumulate aromatic amino acids is very low, and the regulation mechanism of their metabolic pathway is very complex. The traditional mutation breeding methods can only affect the local metabolic pathway or key enzymes, and it is difficult to have a great impact on the global L-tyrosine metabolic flow. In recent years, with the rapid development of metabolic engineering and various advanced biotechnology, it has gradually become a research hotspot to redesign the metabolic pathway of microorganisms to better realize the fermentation of L-tyrosine. The L-tyrosine metabolic engineering bacteria studied more are Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum and Bacillus subtilis. Among them, the synthesis pathway and regulation mechanism of L-tyrosine in Escherichia coli and Helicobacter glutamicum have been studied most and explained most clearly.
L-tyrosine biosynthesis pathway belongs to aromatic amino acid synthesis pathway. Its precursors, Erythropose-4-phosphate (E4P) and phosphoenol pyruvate (PEP), are condensed under the catalysis of DAHP synthetase (DS) to produce 3-deoxy-D-arabinogenosyl-7-phosphate (DAHP), which is also the first rate-limiting step in the biosynthesis of L-tyrosine. In Escherichia coli, DAHP synthetase contains three isozymes, AroG, AroF and AroH, and its expression and activity are subject to feedback inhibition and feedback inhibition of products L-phenylalanine, L-tyrosine and L-tryptophan, respectively. The 7-step reaction from DAHP to branched acid is a common pathway for all aromatic amino acids. Branched acid is the branching point of aromatic amino acid synthesis pathway. One branch pathway is used to synthesize L-tryptophan, and the other part is used to generate p-hydroxyphenylpyruvate (4HPP) under the action of the bifunctional enzyme TyrA of the branching acid mutase (CM) and the prephenate dehydratase (PD), which generates L-tyrosine through the transamination with L-glutamic acid, The expression and enzyme activity of TyrA are also subject to feedback repression and feedback inhibition of the product L-tyrosine.
3. Extraction method
The extraction method was first invented by Braconot in 1820. He extracted glycine and leucine from gelatin sheep color and muscle hydrolysate. After that, Bopp and others gradually hydrolyzed tyrosine and serine in protein. The oldest technology of amino acid production is the white matter hydrolysis extraction method. Protein can be hydrolyzed by enzyme, acid or dexterity, and the final product is amino acid. 6 M hydrochloric acid is usually hydrolyzed at 110 ℃ for 12-24 hours. After removing the excess acid, a mixture of various amino acids is extracted. Finally, the relative purity of amino acids was obtained by using the method of solubility difference or ion exchange resin.
By the 1930s and 1940s, more than 20 kinds of amino acids could be obtained by extraction. The most famous amino acid industry is monosodium glutamate. Today, although most of amino acids can be extracted from various resources, they are not suitable for large-scale production due to high resource cost, low yield and environmental pollution. The extraction method for the production of cardiac tyrosine generally uses natural protein resources as raw materials to separate and extract cardiac tyrosine through hydrolysis, concentration, crystallization, decolorization and other steps. However, because the content of L-tyrosine in the extracted product is low and the yield of the extraction method is low, it is not suitable for large-scale production.
4. Chemical synthesis
Although the chemical synthesis method has been used to synthesize amino acids since the 19th century, it was not until the 1950s that the chemical method was used to synthesize amino acids. This method is to produce amino acids using the technology of organic synthesis and chemical engineering. Its biggest advantage is that it is not limited by the variety of amino acids. In addition to preparing natural amino acids, it can also produce non-natural amino acids, including some amino acids with very special structure, and can be produced on a large scale. However, chemical methods also have disadvantages. The main problem is that the process is complex, and only D and L-type racemates of amino acids can be synthesized. Only after optical resolution can the optically active amino acids be obtained. Up to now, many kinds of amino acids are still produced by chemical synthesis, especially D and L-methionine, which are used in large amounts in feed. The production method is only chemical synthesis, and its output is about several hundred thousand tons/year. In addition, the chemical synthesis method is also used for the large-scale production of medicinal and edible glycine.
L-tyrosine (Tyr) is an important nutritional essential amino acid, which plays an important role in the metabolism, growth and development of human and animals, and is widely used in food, feed, medicine and chemical industries. It is often used as a nutritional supplement for patients with phenylketonuria, as well as a raw material for the preparation of polypeptide hormones, antibiotics, L-dopa, melanin, p-hydroxycinnamic acid, p-hydroxystyrene and other pharmaceutical and chemical products. However, with the discovery of more high-value-added L-tyrosine derivatives such as danshensu, resveratrol, hydroxytyrosol, etc. in organisms, L-tyrosine is increasingly developing towards platform compounds.
1. L-tyrosine is used in medicine to treat hyperthyroidism.
2. L-tyrosine is an important biochemical reagent and the main raw material for the synthesis of peptide hormones, antibiotics, L-dopa and other drugs.
3. L-tyrosine can be used as food additive.
The early production of L-tyrosine mainly depends on protein hydrolysis. However, the protein hydrolysis method has been eliminated due to its shortcomings such as limited material sources, complex process and products, and long cycle. L-tyrosine is mainly produced by enzyme method, microbial fermentation method, extraction method and chemical method.
1. Enzymatic method
Enzymatic method, also known as microbial transformation method, is mainly to convert phenol, pyruvate and ammonia or phenol, L-serine into L-tyrosine by using tyrosine phenol-lyase (TPL, EC 4.1.99.2) in microbial cells. The TPL with high enzyme activity that has been studied extensively mainly comes from the microorganism Erwinia herbicola, Citrobacter intermedium, Citrobacter freundii and Thermophilic bacteria. Lee and Hsiao of Genex Company first used the serine hydroxymethyltransferase of Klebsiella aerogenes and the tyrosine phenol lyase of Erwinia herbicola ATCC 21434 to couple the reaction of synthesizing L-serine with glycine as the substrate and the reaction of synthesizing L-tyrosine with L-serine as the substrate in 1986. Add 0.32% phenol, 0.25 M glycine, 0.5 mM pyridoxal 5-phosphate, 0.056 M in 500 mL reaction system β- Mercaptoethanol, 1.7 mM tetrahydrofolate. When the reaction was started with 37% formaldehyde at pH 7.0 and 37 ℃, L-tyrosine 26.3 g/L was produced after 16 hours, and the conversion rate of glycine reached 61.4%. However, the stability of this process is poor and glycine has a strong inhibitory effect on TPL activity. Considering the disadvantages of poor enzyme activity and stability in the reaction process, the use of DNA shuffling technology to improve the stability of TPL has also attracted attention in recent years. Eugene et al. of KRIBB in South Korea obtained AS6 mutant with catalytic activity increased by three times and half-inactivation temperature increased by 11.2 ℃ through random mutation screening and staggered DNA shuffling of TPL in Symbiobacterium Toebii. The sequencing results showed that there were T129I or T451A mutations in the catalytic active region, and three mutations including A13V, E83K and T407A were greatly helpful to improve the thermal stability. Kim et al ColiBL21 (DE3) overexpressed TPL with improved catalytic activity and thermal stability, and prepared the catalytic crude extract. In a 2.5 L flow plus reactor system, 2.2 M phenol, 2.4 M sodium pyruvate, 0.4 mM pyridoxal 5-phosphate and 4 M ammonium chloride were added in batches and filled with nitrogen above the reaction tank to reduce the oxidation of the substrate. After 30 hours of reaction at 40 ℃, 130 g/L L-tyrosine could be accumulated, and the conversion rate of phenol could reach 94%.
2. Microbial fermentation method
The microbial fermentation method usually uses glycerol, glucose and other biomass carbon sources as raw materials, and accumulates L-tyrosine through the fermentation of excellent microbial strains under appropriate conditions. Early studies often used artificial mutation to select L-tyrosine-producing strains, such as screening L-phenylalanine or L-tryptophan deficiency or anti-feedback inhibition strains. However, the ability of most microorganisms to accumulate aromatic amino acids is very low, and the regulation mechanism of their metabolic pathway is very complex. The traditional mutation breeding methods can only affect the local metabolic pathway or key enzymes, and it is difficult to have a great impact on the global L-tyrosine metabolic flow. In recent years, with the rapid development of metabolic engineering and various advanced biotechnology, it has gradually become a research hotspot to redesign the metabolic pathway of microorganisms to better realize the fermentation of L-tyrosine. The L-tyrosine metabolic engineering bacteria studied more are Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum and Bacillus subtilis. Among them, the synthesis pathway and regulation mechanism of L-tyrosine in Escherichia coli and Helicobacter glutamicum have been studied most and explained most clearly.
L-tyrosine biosynthesis pathway belongs to aromatic amino acid synthesis pathway. Its precursors, Erythropose-4-phosphate (E4P) and phosphoenol pyruvate (PEP), are condensed under the catalysis of DAHP synthetase (DS) to produce 3-deoxy-D-arabinogenosyl-7-phosphate (DAHP), which is also the first rate-limiting step in the biosynthesis of L-tyrosine. In Escherichia coli, DAHP synthetase contains three isozymes, AroG, AroF and AroH, and its expression and activity are subject to feedback inhibition and feedback inhibition of products L-phenylalanine, L-tyrosine and L-tryptophan, respectively. The 7-step reaction from DAHP to branched acid is a common pathway for all aromatic amino acids. Branched acid is the branching point of aromatic amino acid synthesis pathway. One branch pathway is used to synthesize L-tryptophan, and the other part is used to generate p-hydroxyphenylpyruvate (4HPP) under the action of the bifunctional enzyme TyrA of the branching acid mutase (CM) and the prephenate dehydratase (PD), which generates L-tyrosine through the transamination with L-glutamic acid, The expression and enzyme activity of TyrA are also subject to feedback repression and feedback inhibition of the product L-tyrosine.
3. Extraction method
The extraction method was first invented by Braconot in 1820. He extracted glycine and leucine from gelatin sheep color and muscle hydrolysate. After that, Bopp and others gradually hydrolyzed tyrosine and serine in protein. The oldest technology of amino acid production is the white matter hydrolysis extraction method. Protein can be hydrolyzed by enzyme, acid or dexterity, and the final product is amino acid. 6 M hydrochloric acid is usually hydrolyzed at 110 ℃ for 12-24 hours. After removing the excess acid, a mixture of various amino acids is extracted. Finally, the relative purity of amino acids was obtained by using the method of solubility difference or ion exchange resin.
By the 1930s and 1940s, more than 20 kinds of amino acids could be obtained by extraction. The most famous amino acid industry is monosodium glutamate. Today, although most of amino acids can be extracted from various resources, they are not suitable for large-scale production due to high resource cost, low yield and environmental pollution. The extraction method for the production of cardiac tyrosine generally uses natural protein resources as raw materials to separate and extract cardiac tyrosine through hydrolysis, concentration, crystallization, decolorization and other steps. However, because the content of L-tyrosine in the extracted product is low and the yield of the extraction method is low, it is not suitable for large-scale production.
4. Chemical synthesis
Although the chemical synthesis method has been used to synthesize amino acids since the 19th century, it was not until the 1950s that the chemical method was used to synthesize amino acids. This method is to produce amino acids using the technology of organic synthesis and chemical engineering. Its biggest advantage is that it is not limited by the variety of amino acids. In addition to preparing natural amino acids, it can also produce non-natural amino acids, including some amino acids with very special structure, and can be produced on a large scale. However, chemical methods also have disadvantages. The main problem is that the process is complex, and only D and L-type racemates of amino acids can be synthesized. Only after optical resolution can the optically active amino acids be obtained. Up to now, many kinds of amino acids are still produced by chemical synthesis, especially D and L-methionine, which are used in large amounts in feed. The production method is only chemical synthesis, and its output is about several hundred thousand tons/year. In addition, the chemical synthesis method is also used for the large-scale production of medicinal and edible glycine.
