Methionine is one of essential amino acids of human body which has been widely used as feed and food additives and further used as a synthetic raw material for medical solutions and medical supplies. Methionine acts as a precursor of such compounds as choline (lecithin) and creatine and at the same time is used as a synthetic raw material for cysteine and taurine. Methionine can also provide sulfur. S-adenosyl-methionine is derived from L-methionine and plays a certain role in providing methyl group in human body and also is involved in the synthesis of various neurotransmitters in the brain. Methionine and/or S-adenosyl-L-methionine (SAM) inhibits fat accumulation in the liver and artery and alleviates depression, inflammation, liver disease, and muscle pain, etc.
The in vivo functions of methionine and/or S-adenosyl-L-methionine known so far are as follows.
1) It inhibits fat accumulation in the liver and artery promoting lipid metabolism and improves blood circulation in the brain, heart and kidney (J. Hepatol. Jeon B R et al., 2001 March; 34(3): 395-401).
2) It promotes digestion, detoxification and excretion of toxic substances and excretion of heavy metals such as Pb.
3) It can be administered as an anti-depression agent at the dosage of 800-1,600 mg/day (Am J Clin Nutr. Mischoulon D. et al., 2002 November; 76(5): 1158S-61S).
4) It enhances liver functions (FASEB J. Mato J M., 2002 January; 16(1): 15-26) and particularly is effective in the liver disease caused by alcohol (Cochrane Database Syst Rev., Rambaldi A., 2001; (4): CD002235).
5) It has anti-inflammatory effect on bone and joint diseases and promotes joint-recovery (ACP J. Club. Sander O., 2003 January-February; 138(1): 21, J Fam Pract., Soeken K L et al., 2002 May; 51(5): 425-30).
6) It is an essential nutrient for hair. It provides nutrition to hair and thereby prevents hair loss (Audiol Neurootol., Lockwood D S et al., 2000 September-October; 5(5): 263-266).
Methionine can be chemically or biologically synthesized to be applied to feed, food and medicines.
In the chemical synthesis, methionine is mostly produced by hydrolysis of 5-(β-methylmercaptoethyl)-hydantoin. The chemically synthesized methionine has a disadvantage of only being produced as a mixed form of L-type and D-type.
In the biological synthesis, methionine is produced by method using proteins involved in methionine synthesis. L-methionine is biosynthesized from homoserine by the action of the enzyme expressed by such genes as metA, metB, metC, metE and metH. Particularly, metA is the gene encoding homoserine O-succinyl transferase which is the first enzyme necessary for methionine biosynthesis, and it converts homoserine into O-succinyl-L-homoserine. O-succinylhomoserine lyase or cystathionine gamma synthase coded by metB gene converts O-succinyl-L-homoserine into cystathionine. Cystathionine beta lyase coded by metC gene converts cystathionine into L-homocysteine. MetE encodes cobalamine-independent methionine synthase and metH encodes cobalamine-dependent methionine synthase, both of which convert L-homocysteine into L-methionine. At this time, 5,10-methylenetetrahydrofolate reductase coded by metF and serine hydroxymethyltransferase coded by glyA work together to synthesize N(5)-methyltetrahydrofolate providing methyl group necessary for L-methionine synthesis.
L-methionine is synthesized by a series of organic reactions by the above enzymes. The genetic modification on the above proteins or other proteins affecting the above proteins might result in the regulation of L-methionine synthesis. For example, Japanese Laid-Open Patent Publication No. 2000/139471 describes a method of producing L-methionine with the Escherichia sp. of which thrBC and metJ genes on the genome are deleted, metBL is over-expressed and metK is replaced by a leaky mutant. Also, US Patent Publication No. US2003/0092026 A1 describes a method using a metD (L-methionine synthesis inhibitor) knock-out microorganism which belongs to Corynebacterium sp. US Patent Publication No. US2002/0049305 describes a method to increase L-methionine production by increasing the expression of 5,10-methylenetetrahydrofolate reductase (metF).
US Patent No. US2005/0054060A1 describes the method of preparing L-methionine producing microorganism using cystathionine synthase (O-succinylhomoserine lyase) mutant. This cystathionine synthase mutant can produce homocysteine or methionine directly from H2S or CH3SH instead of cysteine. In this method, mutant cystathionine synthase is directly introduced into a cell and participated in the intracellular methionine biosynthesis procedure. In this method, cystathionine synthase reaction is not very efficient due to the use of intracellular methionine biosynthesis pathway. Also, the high toxicity of H2S or CH3SH to the cells reduces the effectiveness of this method. In our experiment, we also found that the that substrate specificity of cystathionine synthase to CH3SH is very low compared to succinylhomoserine lyase derived from Pseudomonas or Chromobacterium sp.
According to the previous reports, cystathionine synthase tend to produce various products by reaction with various substrates. Cystathionine synthase mediates the interaction between homocysteine and O-succinyl homoserine to produce homolanthionine with high efficiency (J. Bacteriol (2006) vol 188: p 609-618). The cystathionine synthase in a cell can interact with various methionine precursors and can produce various byproducts with high efficiency. Therefore, overexpression of Cystathionine synthase can make lower the reaction efficiency due to the higher production of byproduct.
The methionine produced by the conventional biological method is L-type, which has advantages but the production amount is too small. This is because the methionine biosynthetic pathway has very tight feed-back regulation systems. Once methionine is synthesized to a certain level, the final product methionine inhibits the transcription of metA gene encoding the primary protein for initiation of methionine biosynthesis. Over-expression of metA gene itself cannot increase methionine production because the metA gene is suppressed by methionine in the transcription stage and then degraded by the intracellular proteases in the translation stage (Dvora Biran, Eyal Gur, Leora Gollan and Eliora Z. Ron: Control of methionine biosynthesis in Escherichia coli by proteolysis: Molecular Microbiology (2000) 37(6), 1436-1443). Therefore, many of previous patents were focused on how to free the metA gene from its feed-back regulation system (WO2005/108561, WO1403813).
When methionine is produced in biological system, produced methionine is converted to S-adenosylmethionine by S-adenosylmethionine synthase in the methionine degradation pathway. S-adenosylmethionine synthase cannot be deleted because S-adenosylmethionine is an essential substance in cells. According to the previous patents, the gene encoding S-adenosylmethionine synthase was mutated to have low activity to increase the methionine production (WO2005/108561).