Methionine production by fermentation - iBrarian

Biotechnology Advances 23 (2005) 41 – 61 www.elsevier.com/locate/biotechadv

Research review paper

Methionine production by fermentation Dharmendra Kumara,*, James Gomesb a

Department of Biotechnology, Sun Pharma Advanced Research Centre, Vadodara-390 020, India b Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, New Delhi-110016, India Received 11 August 2004; received in revised form 24 August 2004; accepted 24 August 2004 Available online 12 October 2004

Abstract Fermentation processes have been developed for producing most of the essential amino acids. Methionine is one exception. Although microbial production of methionine has been attempted, no commercial bioproduction exists. Here, we discuss the prospects of producing methionine by fermentation. A detailed account is given of methionine biosynthesis and its regulation in some potential producer microorganisms. Problems associated with isolation of methionine overproducing strains are discussed. Approaches to selecting microorganism having relaxed and complex regulatory control mechanisms for methionine biosynthesis are examined. The importance of fermentation media composition and culture conditions for methionine production is assessed and methods for recovering methionine from fermentation broth are considered. D 2004 Elsevier Inc. All rights reserved. Keywords: Fermentation; Methionine production; Metabolic regulation; Strain improvement; Corynebacterium

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . Selection of microorganisms for the production of Methionine biosynthesis in microorganisms. . . . Metabolic regulation of methionine biosynthesis .

. . . . . . . . l-methionine . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +91 265 2341400; fax: +91 265 339103. E-mail address: [email protected] (D. Kumar). 0734-9750/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2004.08.005

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D. Kumar, J. Gomes / Biotechnology Advances 23 (2005) 41–61

5. Strain improvement for overproduction of methionine . . . . . . . . 6. Production of methionine by auxotrophic and regulatory mutants . . 7. Roles of trans-sulfuration and reverse trans-sulfuration in methionine 8. Media composition and culture conditions . . . . . . . . . . . . . . 9. Recovery of methionine from fermented broth . . . . . . . . . . . . 10. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . production . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Methionine is an essential amino acid that is required in the diet of humans and livestock. Plant proteins are frequently deficient in methionine and consequently an exclusively vegetable diet may fail to meet nutritional requirements. Methionine deficiency has been linked to development of various diseases and physiological conditions including toxemia, childhood rheumatic fever, muscle paralysis, hair loss, depression, schizophrenia, Parkinson’s liver deterioration, and impaired growth (Rose, 1938). Deficiencies can be overcome by supplementing the diet with methionine and, therefore, methionine is of significant interest (Parcell, 2002). In April 2000, the Complementary Medicines Evaluation Committee (CMEC) recommended that l-methionine is suitable for use as an ingredient in therapeutics and does not require any substance-specific restrictions on its use. Methionine is extensively used in the poultry and feedstock industry (Tabor et al., 1958; Neuvonen et al., 1985; Funfstuck et al., 1997; Campbell, 2001). Because fermentation processes have been able to inexpensively provide many other amino acids, there is a significant interest in developing a microbial process for commercial production of methionine (Pham et al., 1992; Umerie et al., 2000; Odunfa et al., 2001). Currently, methionine is produced either by chemical synthesis or by hydrolyzing proteins. These processes are expensive. Chemical synthesis produces a mixture of d- and lmethionine (Mannsfeld et al., 1978; Leuchtenberger, 1996) whereas hydrolysis of proteins leads to a complex mixture from which methionine must be separated. Chemically produced racemic mixture of methionine isomers can be resolved using continuous flow immobilized enzyme bioreactors containing fungal aminoacylases (Tosa et al., 1967); nevertheless, the chemical production of the racemic mixture is undesirable as it requires on hazardous chemicals such as acrolein, methyl mercaptan, ammonia and cyanide (Fong et al., 1981). Biologically active l-methionine can be produced either by enzymatic synthesis (bioconversion of precursors), or by submerged fermentation using microorganisms. lmethionine has been produced enzymatically by the stereospecific cleavage of N-acyl-dlmethionine using amino acylases (Chibata et al., 1957). Tokuyama and Hatano (1996) inserted the gene for N-acylamino acid racemase from Amycolaptosis sp. TS-1-60 in Escherichia coli and reported continuous production of l-methionine in high yield. Yamashiro et al. (1988) reported bioconversion of dl-5-substituted hydantoins into lform of amino acids including methionine by Bacillus brevis and its mutants. Wagner et al. (1996) reported bioconversion of dl-5-(2-methylthioethyl)-hydantoin into methionine with 90% yield by resting cells of the mutant strain DSM 9771. Morinaga et al. (1982a,b)


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used resting cells of ribulose-monophosphate pathway-type methylotroph strain OM 33 and serine pathway-type methylotroph Pseudomonas FM 518 to catalyze the formation of methionine from dl-homocysteine and methanol. Although the existing enzymatic processes achieve good yields, they require expensive substrates. This is motivating research in methionine production by fermentation. The discovery of glutamic acid producing bacteria by Kinoshita et al. (1957) eventually led to fermentation processes for producing various other amino acids. Since then, a number of microorganisms capable of producing amino acids have been isolated and the production of amino acids has become an important aspect of industrial microbiology. Amino acids such as lysine, threonine, isoleucine, and histidine have been produced successfully by fermentation (Fan et al., 1988; Leuchtenberger, 1996; Okamoto and Ikeda, 2000a; Okamoto et al., 2000b; Kircher and Pfefferle, 2001; Hermann, 2003). Attempts have been made to overproduce biologically active l-methionine using fermentation (Kase and Nakayama, 1975a; Nakayama et al., 1978; Roy et al., 1984, 1989; Mondal et al., 1994, Kumar et al., 2003), but no methionine fermentation has been commercialized. This review discussed various aspects of microbial production of methionine, to facilitate development of microbial production of this amino acid.

2. Selection of microorganisms for the production of L-methionine To successfully establish a commercially viable process for microbial production of methionine, a high producer organism must be found or generated. Wildtype strains are not usually capable of producing significant amount of methionine because its biosynthesis is highly regulated (Rowbury and Woods, 1961; Herrmann and Somerville, 1983). Methionine fermentation is significantly different than the other conventional fermentations such as

Fig. 1. Regulation of methionine biosynthesis in Corynebacterium and Brevibacterium (Tosaka and Takinami, 1986). Blue arrows show inhibition and dotted arrows represent repression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Regulation of methionine biosynthesis in E. coli (Tosaka and Takinami, 1986). Blue arrows show inhibition and dotted arrows show repression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

those for ethanol, lactic acid and citric acid. Unlike ethanol and lactic acid that are waste products of pathways used to generate adenosine tri-phosphate (ATP), synthesis of methionine requires energy (ATP). Therefore, overproduction of methionine is tremendously wasteful to the microorganisms and only the methionine needed for growth is produced. It has been observed that strains of Corynebacterium and Brevibacterium have much simpler regulatory mechanisms for methionine production (Fig. 1) than does E. coli in which all the enzymes are inhibited or repressed by the end products (Tosaka and Takinami, 1986). This is presumably because these organisms evolved in an environment that was poor in amino acids. If an organism lives in amino acid scarce environment, the major function of the regulation of amino acid biosynthesis is then to adjust its rate of production in response to the growth rate of the organism. Thus, there is no need to adjust the ratios of the various amino acids produced in the cell because when excess methionine is produced, excess threonine, lysine and isoleucine are likely produced because the same metabolic pathway is involved in their production. All major regulation of such a pathway can be achieved by a system in which only few of the end products inhibit the first common enzyme as shown in Fig. 2. Consequently, it would be better to select microorganisms having comparatively simple regulatory mechanisms for producing methionine. In addition to Corynebacteria and Brevibacteria, more complex microorganisms such as certain Lactobacilli and yeasts have been evaluated for producing methionine and lysine (Odunfa et al., 2001). Mondal et al. (1996) reported several methionine-producing microorganisms.

3. Methionine biosynthesis in microorganisms Methionine, lysine, threonine and isoleucine are the members of aspartate family of amino acids (Fig. 3). The pathways for the biosynthesis of amino acids of this family have


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Fig. 3. Methionine biosynthesis in Corynebacterium glutamicum (Ru¨ckert et al., 2003). [1] Aspartate kinase, [2] aspartaldehyde dehydrogenase, [3] homoserine dehydrogenase, [4] homoserine O-acetyltransferase, [5] Oacetylhomoserine (thiol)-lyase (cystathionine g-synthase), [6] cystathionine g-lyase, [7] homoserine Smethyltransferase (vitamin B12 independent or metH-encoded, vitamin B12 dependent), [8] homoserine kinase, [9] threonine synthase, [10] O-acetylhomoserine sulfhydrylase, [11] serine hydrooxymethyle transferase.

been reported by Stadtman et al. (1961), Yugari and Gilvarg (1962), Rowbury (1964c), Patte and Bros (1967), Umbarger (1969) and Morinaga et al. (1996). Details of methionine biosynthesis have been studied (Flavin, 1975; Kinoshita, 1985; Jetten and Sinskey, 1995; Sahm et al., 1995; Malumbres and Martin, 1996; Hwang et al., 1999). The complete metabolic pathway for methionine biosynthesis in E. coli was reported by Wijesundra and Woods (1962) and Rowbury and Woods (1961, 1964a,b), Rowbury, 1964c. Flavin et al. (1964) detailed methionine biosynthesis in Salmonella typhimurium. Regulation of methionine biosynthesis in Corynebacterium was reported by Kase and Nakayama (1975b) and in Brevibacterium flavum by Ozaki and Shiio (1982). Auger et al. (2002) discussed methionine biosynthesis and its regulation in Bacillus subtilis. Ru¨ckert et al. (2003) elucidated the pathway for the biosynthesis of l-methionine in Corynebacterium glutamicum using genome sequences (Fig. 3). The pathways of methionine biosynthesis in various microorganisms have many common features. Even though microorganisms use different biosynthetic routes, most bacteria and fungi synthesize methionine. Typically, aspartate is converted to 4-phosphoaspartate by aspartate kinase and then oxidized by aspartaldehyde dehydrogenase to form aspartate semi-aldehyde. The latter is oxidized by homoserine dehydrogenase to produce homoserine. Alternatively, aspartate semi-aldehyde is converted to dihydropicolinate by dihydropicolinate-synthase, leading to the formation of lysine. From homoserine, one metabolic path leads to methionine while another leads to threonine and subsequently to

46


isoleucine. Homoserine undergoes condensation with succinyl CoA to produce O-succinyl homoserine. The enzyme responsible for this reaction in E. coli is homoserine Osuccinyltransferase. Most bacteria produce O-succinylhomoserine as an intermediate. In contrast, most fungi and some bacteria (e.g. Bacillus and Corynebacterium) produce Oacetylhomoserine instead of O-succinylhomoserine (Flavin, 1975). Formation of methionine from O-acetyl homoserine may occur in two possible metabolic pathways. Either homocysteine forms through cystathionine (e.g. in some enteric bacteria and fungi such as Neurospora crassa), or acetyl homoserine is directly converted to homocysteine by O-acetyl homoserine (thiol)-lyase (Kerr and Flavin, 1970). Morinaga et al. (1982a) made similar observations for the facultative methylotroph Pseudomonas FM 518. This microorganism possesses activities of both h-cystathioninase and O-acetyl homoserine sulfhydrilase. Cystathionine is synthesized from O-succinyl homoserine and cysteine using the enzyme cystathionine g-synthase (Flavin et al., 1964). This step is reversible and requires pyridoxal phosphate as a cofactor. Cystathionine is then cleaved to homocysteine, pyruvate and ammonia by cystathionine-h-lyase, a pyridoxal phosphate-dependent enzyme (Rowbury and Woods, 1964a). The methionine biosynthetic pathway in B. flavum involves formation of O-acetylhomoserine from homoserine by the action of homoserine O-acetyltransferase and direct formation of homocysteine from O-acetylhomoserine by the O-acetylhomoserine sulfhydrylase (AHS) reaction (Ozaki and Shiio, 1982). Hwang et al. (2002) reported the presence of two parallel pathways, the transsulfuration pathway and direct sulfhydrylation pathway, for methionine biosynthesis in C. glutamicum and related coryneform bacteria such as C. lactofermentum and B. flavum. In the case of trans-sulfuration, cysteine serves as the sulfur donor for the reaction with Oacetyl-l-homoserine resulting in l-cystathionine as described for E. coli (Smith, 1971). In the case of direct sulfhydrylation, inorganic sulfide is used for the formation of O-acetyl-lhomoserine as described for Leptospira meyeri (Belfaiza et al., 1998). Cysteine and homocysteine can be synthesized directly from reduced sulfur, or by the interconversion of these two metabolites. Thiolation pathways directly incorporate sulfide into O-acetylserine or O-acetylhomoserine to produce cysteine or homocysteine, respectively. These reactions are catalyzed by an O-acetylserine thiolyase (Kredich, 1987), or by an O-acetylhomoserine thiolyase (Yamagata, 1989). Saccharomyces cerevisiae (Thomas and Surdin-Kerjan, 1997) and bacteria such as B. flavum (Ozaki and Shiio, 1982) and L. meyeri (Belfaiza et al., 1998) can synthesize homocysteine by thiolation. The trans-sulfuration pathways allow the interconversion of homocysteine and cysteine via the intermediary formation of cystathionine. The synthesis of homocysteine from cysteine is the only means of trans-sulfuration in enteric bacteria (Greene, 1996). In E. coli, this requires the sequential action of cystathionine gsynthase, the metB gene product, and cystathionine h-lyase, the metC gene product (Duchange et al., 1983). Homocysteine on methylation by homocysteine S-methyltransferase leads to the formation of methionine (Kerr and Flavin, 1970; Bourhy et al., 1997; Thomas and Surdin-Kerjan, 1997). The methylation of homocysteine by homocysteine S-methyltransferase may be vitamin B-12 dependent or independent, but 5-methyltetrahydrofolate (a polyglutamate derivative) acts as a methyl donor in both the cases (Kung et al., 1972).


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4. Metabolic regulation of methionine biosynthesis All microorganisms have mechanisms for regulating the quantities and types of enzymes so that only the needed amounts of amino acids are synthesized. This regulatory mechanism must be inactivated or otherwise modulated to ensure the production of a target amino acid in large amounts. Metabolic regulation of biosynthesis of amino acids of aspartate family has been studied (Miyajima and Shiio, 1973; Kase and Nakayama, 1975b; Herrmann and Somerville, 1983). Aspartate kinase (EC 2.7.2.4) is the first enzyme of the metabolic pathway that produces aspartate family of amino acids. This enzyme catalyses the phosphorylation of aspartate. In E. coli, each product of this metabolic pathway inhibits or represses the first enzyme of the pathway (Fig. 2), but the regulatory mechanism is such that one product does not shut down the production of other products. This is because there are three distinct aspartate kinases in E. coli for catalyzing the same reaction and they are affected by different amino acids. Aspartate kinase I is inhibited by threonine and its synthesis is repressed by threonine and isoleucine. Aspartate kinase II is inhibited or repressed by methionine and aspartate kinase III is inhibited or repressed by lysine. The regulation of this branched pathway is very complicated, because it requires a system in which an excess of one product does not shut down the entire pathway (Herrmann and Somerville, 1983). The reduction of aspartate-semialdehyde to homoserine is catalyzed by two distinct homoserine dehydrogenases (EC 1.1.1.3). Synthesis of homoserine dehydrogenase I is repressed by threonine and isoleucine, and its activity is inhibited by threonine whereas homoserine dehydrogenase II is repressed by methionine (Figs. 1 and 2). In addition, E. coli is able to adjust to the presence of an unbalanced mixture of the products of the pathway. If it encounters an excess of methionine but low levels of lysine, threonine and isoleucine, then regulating aspartate kinase alone will not lead to the proper ratio of methionine to the other three amino acids. Thus, each amino acid usually controls the first enzyme of its own particular branch. In contrast, Brevibacterium and Corynebacterium sp. have much simpler regulation of aspartate family amino acid synthesis. These microorganisms have only one aspartate kinase that is inhibited by lysine and threonine in a concerted manner. Efficient feedback inhibition of aspartate kinase therefore requires both lysine and threonine to be present (Fig. 1). When lysine and threonine are present simultaneously at 1 mM, they inhibit aspartate kinase by 94%, whereas each amino acid present alone at 1 mM, inhibits aspartate kinase only by 12–20% (Tosaka and Takinami, 1986). Studies on regulatory aspects of methionine biosynthesis in C. glutamicum show that exogenous methionine is a potent repressor of the synthesis of some enzymes of the pathway (Kase and Nakayama, 1974, 1975c). The enzyme catalyzing the production of homoserine ester is subject to feedback inhibition by methionine and S-adenosylmethionine in E. coli and B. subtilis (Brush and Paulus, 1971; Greene, 1996). However, Archer et al. (1991) reported that homoserine dehydrogenase (HD) is allosterically inhibited by lthreonine in E. coli, B. subtilis and C. glutamicum. The methionine biosynthesis is also regulated at the transcriptional level. In C. glutamicum, most genes coding for the enzymes of the l-methionine biosynthesis are well characterized (Grossmann et al., 2000; Kim et al., 2001; Hwang et al., 2002; Ru¨ckert et al., 2003). Two regulators are involved in this control in E. coli, the MetJ repressor and the

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MetR activator. The MetJ repressor, interacting with S-adenosylmethionine, binds to the Met box sequences and represses the transcription of most of the met genes. MetR stimulates the expression of the metE and metH genes that encode the methionine synthases. Homocysteine markedly enhances the MetR activation of metE expression (Weissbach and Brot, 1991; Greene, 1996). Table 1 lists some of the enzymes, the encoding genes, and their regulators involved in methionine biosynthesis. A number of genes and operons in B. subtilis that are thought to be involved in methionine or cysteine biosynthesis contain a highly conserved sequence upstream of their coding sequence (Grundy and Henkin, 1998) suggesting that regulation is controlled at the level of premature termination of transcription. Rey et al. (2003) reported that the putative transcriptional repressor McbR is involved in the regulation of the metabolic network directing the synthesis of l-methionine in C. glutamicum. Rowbury (1965) reported that resistance to norleucine (analogue of methionine) in microorganism is associated with a failure of methionine to repress any of the biosynthetic enzymes, suggesting that a mutation to resistance alters a single regulator site which, in a repressible strains controls the whole biosynthetic sequence presumably by the synthesis of a diffusible repressor. In some organisms, S-adenosylmethionine (SAM) or its derivatives rather than methionine itself are likely to be the co-repressors of the methionine biosynthesis. SAM represses enzymes involved in methionine biosynthesis. The participation of SAM in repression and inhibition indicates that SAM is likely an end Table 1 Regulation of enzymes involved in methionine biosynthesis Enzyme

Activity inhibited by

Corresponding gene

Gene expression regulated by

Aspartate kinase II (E. coli and S. tiphimurium) Aspartaldehyde dehydrogenase

Methionine

MetL

Methionine

–

Asd

Aspartate kinase I in (Bacillus megaterium) Homoserine dehydrogenase (E. coli and S. tiphimurium) Homoserine-O-succinyl transferase (E. coli and S. tiphimurium) Homoserine-O-acetyltransferase (Brevibacterium flavum) Cystathionine-g-synthase h-cystathioninse Cystathionine-h-lyase O-acetylhomoserine(thiol)-lyase Homocysteine S-methyltransferase (vitamin B12 independent) Homocysteine S-methyltransferase (vitamin B12 dependent) Methylenetetrahydrofolate reductase (vitamin B12 dependent) Transacetylase in yeast

Methionine+lysine

–

Lysine, methionine, threonine Lysine+threonine

Methionine

MetL

Methionine

Methionine, S-adenosylmethionine Methionine, S-adenosylmethionine – – – – –

MetA

–

MetA

Methionine

MetB MetC

Methionine

– MetE

Methionine S-adenosyl-methionine –

–

MetH

Vitamin B12

–

MetH

S-denosylmethionine

S-adenosyl-methionine

–

Methionine


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product in the methionine biosynthesis in Corynebacterium (Kase and Nakayama, 1975b). Synergistic feedback inhibition of the first enzyme of methionine biosynthesis is also found in Bacillus. It has been reported that methionine and SAM inhibit homoserine acetyltransferase of Bacillus polymyxa and each inhibitor binds to a different specific site that is distinct from the active site (Wyman and Paulus, 1975). In S. cerevisiae, SAM inhibits homoserine acetyltransferase, the first enzyme of the metabolic pathway of methionine biosynthesis, alone rather than in conjunction with methionine. In E. coli, the genes of the methionine regulon are distributed throughout the chromosome (Bachmann, 1983). The regulatory functions of S-adenosylmethionine as corepressor and of the metJ gene product as aporepressor have been confirmed by studies of in vitro expression of some of the E. coli methionine biosynthesis genes (Shoeman et al., 1985a,b). However, none of the metJ and metK isolated regulatory mutants were fully decontrolled for methionine production (Su et al., 1970; Greene et al., 1973). Patte and Boy (1972) reported that in E. coli, the enzyme aspartaldehyde dehydrogenase is of only one kind and is multi-valently repressed by lysine, threonine and methionine. Urbanowski et al. (1987) have identified a regulatory locus, the metR, required for the expression of metE and metH genes. However, metE gene has been shown to auto-regulate its own synthesis in in vitro studies while metR protein stimulates its in vitro expression (Cai et al., 1989). The metR gene is a trans-activator of the expression of metE gene and metR gene is under autogenous regulation and is repressed by metJ protein (Maxon et al., 1989). Two of the enzymes involved in the terminal steps of methionine biosynthesis are repressed in a non-coordinated manner by both vitamin B12 and methionine in E. coli (Kung et al., 1972). Nakamori et al. (1999) derived l-methionine analogues resistant mutants of E. coli producing 910 mg/l and reported that only one point mutation in the metJ gene occurred in the l-methionine producing mutants. They cloned the metJ gene coding for the E. coli met repressor metJ protein and found single amino acid substitution (wildtype Ser–Asn) at position 54 in four independent l-methionine producing mutants. When wildtype metJ gene were introduced into the mutants having mutant metJ gene, the level of the enzyme synthesis and l-methionine production in the tranformants reverted to wildtype levels (Nakamori et al., 1999).

5. Strain improvement for overproduction of methionine Success of fermentation processes depends on the potential of the producing strain. As no wildtype microorganism overproduces methionine, a mutant with genetically altered regulatory mechanism must be developed for producing this amino acid (Yamada et al., 1982; Mondal and Chaterjee, 1994). Strain improvement by classical mutagenesis techniques is well established and widely used to isolate amino acid overproducers (Rowlands, 1984; Sharma, 2002). Screening procedures have been designed to allow for isolation of the overproducer mutants. Amino acid analogues can effectively function as true feedback inhibitors without participating in other functions in the cell (Morinaga et al., 1982a,b; Chattopadhyay et al., 1995a). Mutants resistant to methionine analogues have altered and deregulated enzymes that are not sensitive to feedback inhibition and repression. Such mutants in the absence of

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analogues can synthesize methionine in excess and eventually excrete it into the fermentation broth (Kase and Nakayama, 1975a; Roy et al., 1984; Mondal et al., 1994). Kumar et al. (2003) described a method for isolating analogue-resistant mutants from the wildtype strain. Resistance to analogues can originate in any of the following ways: (1) mutation in an operator, resulting in de-repression of biosynthetic enzymes; (2) mutation in a structural gene of a biosynthetic enzyme subjected to end product inhibition, resulting in loss of feedback control; (3) mutation in structural gene of an amino acyltransfer RNA synthase, resulting in discrimination between amino acid and the analogue; and (4) mutation in the structural gene of a permease for the amino acid, resulting in reduced ability to take up the analogue. Auxotrophic mutants with resistance to analogues are widely used for commercial production of amino acids. Penicillin enrichment can be used for isolating auxotrophic mutants (Hopwood, 1970). This method can be modified to isolate a particular auxotroph. After mutagenesis cells are allowed to grow in a complete medium (i.e. a medium that contains all the requisite amino acids) to increase the number of auxotrophs. The biomass is then recovered by centrifugation, washed with phosphate buffer and grown in a minimal medium that contains 25–50 Ag/ml of penicillin. Prototrophs will grow but eventually die because of leaky cell walls produced in the presence of penicillin. Auxotrophs will not grow but remain alive. The recovered live cells can now be grown in minimal media containing particular amino acid to isolate the auxotrophic mutants. It may be possible to obtain overproduction of lysine simply by cutting out the pathway branches leading to the other amino acids (homoserine auxotroph). Regulatory mutants of Corynebacterium are reported to produce 54 g/l lysine (Hirao et al., 1989), while auxotrophic regulatory mutant produced 76 g/l lysine (Hadj-Sassi et al., 1990). Threonine production of more than 70 g/l by auxotrophic regulatory mutants has been reported (Yamada et al., 1987; Kino et al., 1993). It is evident from these observations that auxotrophic regulatory mutants are more useful than other mutants for the production of methionine. Undesirable inhibition can be eliminated through such mutations to greatly increase the yield of the target amino acid. Presumably, lysine and threonine dual auxotrophs with resistance to methionine analogues will overproduce methionine because in such mutants there will be neither undesirable inhibition of aspartate kinase and homoserinedehydrogenase by lysine and threonine nor a wastage of carbon for the production of these metabolites. Improvement of strain to increase its productivity depends on detailed information of metabolic pathways, their regulation and control. Genetic engineering is now a powerful alternative to mutagenesis for obtaining overproducers (Sahm et al., 1995, 2000). Furthermore, use of genetic engineering eliminates the need for isolating the useful mutant from the many nonuseful ones that are generated by classical mutagenesis (Kalinowski et al., 2003). Methionine production by microorganism can be enhanced by modifying the pathway of methionine biosynthesis and altering its regulatory mechanism. This involves transforming or transducing the host (Corynebacterium or Brevibacterium sp.) with homoserine-activating enzymes, homoserine acetyltransferase or homoserine succinyltransferase gene fragment and a sulfur incorporating enzyme (e.g. O-succinylhomoserine-


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(thiol)-lyase or O-acetylhomoserine-(thiol)-lyase) gene fragment. The transformants are then recovered and an exogenous S-source is provided to the cells for producing methionine (Genencor, 1993). Several genes from biosynthetic pathways leading to aspartate-derived amino acids in C. glutamicum have been cloned, analyzed and identified by heterologous complementation of E. coli mutants and, occasionally, in the homologous system by conferring an amino acid analogue resistance. By cloning and expressing the various genes of the llysine pathway in C. glutamicum, an increase in the flux of l-aspartate semialdehyde to l-lysine has been obtained in strains with increased dihydrodipicolinate synthase activity. By combined overexpression of deregulated aspartate kinase and dihydrodipicolinate synthase, the l-lysine secretion was increased by 10–20% (Sahm et al., 1995). Yield of methionine can also be increased similarly by metabolic flux engineering and cloning techniques. A poly-methionine DNA fragment containing plasmid that was transformed into a parent yeast cell CB89, enhanced the methionine production in comparison to simple plasmid transfer (Halasz et al., 1995). Similarly, E. coli containing a DNA inversion gene showed hypersecretion of phenylalanine, methionine and tyrosine (Fotheringham, 1994). Sahm et al. (1995) reported that C. glutamicum strains do not secrete amino acids via passive diffusion but via specific active carrier systems. This secretion carrier has a strong influence on the overproduction of amino acids. Thus, for the construction of really good overproducers by gene engineering, both the synthesis and export machinery must be modified.

6. Production of methionine by auxotrophic and regulatory mutants Commercial production of most amino acids uses auxotrophic, regulatory or auxotrophic regulatory mutants. All regulatory mechanisms such as feedback inhibition and repression require the end product to operate. If one of the steps in the biosynthesis of the amino acid is blocked, the regulatory system will not work and the cell will produce the intermediate in excess causing accumulation before the blocked step. Auxotrophic mutants are generally useful for the production of intermediate metabolite of the straight chain pathway but they can also be useful for the production of branched pathway amino acids such as lysine, threonine and methionine. Although, mostly, analogue resistant regulatory mutants have been assessed for producing methionine, it would be better to use auxotrophic regulatory mutants because then methionine itself will not inhibit or repress its own production. Mondal et al. (1994) reported that a methionine-resistant mutant of B. heali produced 13 mg/l methionine, whereas lysine and threonine auxotrophic regulatory mutants of the same bacterium produced 25.5 g/l methionine (Mondal et al., 1994). Regulatory mutants are generally used for producing amino acids. Roy et al. (1984) and Kumar et al. (2003) used a multi-methionine analogues resistant mutant (regulatory mutant) for the production of methionine. Such overproducer mutants for methionine can be isolated using analogues of methionine. Analogues inhibit growth of the wildtype strain in minimal media. In the past, the prevailing view was that these analogues inhibited growth by incorporating into proteins and thereby producing non-functional proteins. This

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may occur in some instances, but in vast majority of the cases, the major cause of inhibition appears to be that the analogues mimic the way the amino acid regulates its own production. Thus, analogues may bind to the allosteric or the product site of the enzyme or may bind effectively to the repressor and consequently shut down the pathway for the synthesis of that particular amino acid. Analogues inhibit growth by starving the cell of the particular amino acid. Therefore, amino acid analogues act as pseudo-feedback inhibitors or repressors, thereby inhibiting or repressing the synthesis of the corresponding amino acid. Only mutants having resistance to analogues may overproduce corresponding amino acid. These mutants are able to resist the analogues either because of an alteration in the structure of the enzyme or an alteration in the enzyme formation system. In the case of methionine, analogues such as ethionine, seleno-methionine, norleucine, and methionine hydroxamate have been used to develop methionine-overproducing strains (Kase and Nakayama, 1975a,b,c; Mondal et al., 1994). Table 2 lists the methionine-producing microorganisms. Methionine overproduction by ethionine resistant mutants has been reported for S. cerevisiae (Cherest et al., 1973), S. lipolytica (Morzycka et al., 1976) and an n-paraffin utilizing yeast, Candida petrophilum (Komatsu et al., 1974). A methylotrophic yeast, C. boidinii no. 2201, produced 16.02 mg/g-DCW of total methionine (Tani et al., 1988). Halasz et al. (1988) also reported a methionine-rich yeast for use as single-cell protein.

Table 2 Methionine production by some methionine analog resistant regulatory mutants Microorganism

Genetic marker

Methionine yield (g/l)

Reference

Bacillus megaterium B71 Bacillus mageterium Bacillus brevis AJ 122299 Brevibacterium heali Corynebacterium lilium NTE 99 Corynebacterium glutamicum

–

4.5 4.2 4.26 1.3 4.074 2.0

Roy et al. (1984) Roy et al. (1984) Yamashiro et al. (1988) Mondal and Chaterjee (1994) Sharma and Gomes (2001) Kase and Nakayama (1975a,b,c) Tani et al. (1988)

Candida biodini E-500-78b

ethR – Thr, met analogR ethR

Candida biodini E500-78

ethR

E. coli E. coli K12 E. coli JM109 TN1 Kluyveromyces lactis IPU126 M. glutamicus Methylomonas sp. Neurospora crassa OM 33 Pseudomonas FM 518 Pseudomonas FM 518 Saccharomyces cerevisiae Serratia sp.

ethR nleR ethR EMS MNNGR ethR ethR MNNGR ethR MNNGR ethR EthR –

8.80 mg/g of dry cell 6.02 mg/g of dry cell 2.0 2.0 0.91 14.2c 3.0 0.42 2.0 0.8 0.8 0.33 0.78

Tani et al. (1988) Rowbury (1965) Chattopadhyay et al. (1995a,b) Nakamori et al. (1999) Kitamoto and Nakahara (1994) Banik and Majoomdaar (1974) Yamada et al. (1982) Metzenberg et al. (1964) Yamada et al. (1982) Morinaga et al. (1982a,b) Morinaga et al. (1982a,b) Brigidi et al. (1988) Ghosh and Banerjee (1986)


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Ethionine resistant mutant of a methylotrophic bacterium was reported to produce 420 mg/ l methionine under optimized conditions (Yamada et al., 1982). A dl-ethionine-resistant mutant ER 108, carrying a mutation to chloramphenicol resistance, and protoplasts obtained from it were fused with protoplasts from antibiotic sensitive S. cerevisiae X2928 carrying six auxotrophies. The resulting fusants maintained four auxotrophies and were capable of overproducing l-methionine (Brigidi et al., 1988). A multi-analogue resistant and threonine auxotrophic mutant of C. glutamicum ESLMR-724 produced 2 mg/ml methionine in a medium containing 10% glucose (Kase and Nakayama, 1975a,b,c). Ghosh and Banerjee (1986) reported that a hydrocarbon utilizing Serratia marcescens var kiliensis produced 1.68 g/l glutamic acid and 0.78 g/l methionine under optimized culture conditions in a synthetic medium with hydrocarbon as the sole carbon-source. A mutant of E. coli K12, resistant to methionine (norleucine, ethionine and a-methylmethionine) and threonine analogue (a-amino-h-hydroxy valeric acid) produced 2 g/l of both methionine as well as threonine (Chattopadhyay et al., 1995a). Chattopadhyay et al. (1995b) also reported production of 1 g/l of both methionine as well as threonine using ethionine and 5-bromouracil resistant strain of E. coli K-12. Nakamori et al. (1999) reported 910 mg/l l-methionine production by a methionine analogue resistant mutant strain TN1 of E. coli JM109.

7. Roles of trans-sulfuration and reverse trans-sulfuration in methionine production Although attempts have been made to develop a process for producing methionine by fermentation, no remarkable breakthroughs have been reported. Presumably, the methionine analogues resistant mutants will overproduce methionine, but in the presence of excess of intracellular methionine the reverse trans-sulfuration pathway becomes active to form cysteine. Cysteine itself is a regulatory metabolite and no wildtype strain is known to overproduce it (Wada et al., 2002). In order to overproduce cysteine, a microorganism should have serine acetyltransferases that have been desensitized to feedback inhibition or repression (Nakamori et al., 1998; Tagaki et al., 1999). It is well known that in bacteria and fungi, cysteine is synthesized either via serine Oacetyltransferase and O-acetylserine sulfhydrylase, or by the reverse trans-sulfuration pathway starting from homocysteine (Vermeij and Kertesz, 1999). If the medium is rich in methionine, the microorganism will prefer to synthesize cysteine from methionine by the reverse trans-sulfuration pathway (to conserve energy) resulting in reduced methionine production. Kumar et al. (in press) observed that cysteine supplementation of the fermentation medium increased methionine yield from 2.34 to 3.39 g/l using a regulatory mutant of Corynebacterium lilium. Unfortunately, cysteine supplementation of the fermentation medium is unlikely to be economically feasible. Therefore, overproduction of methionine will need to rely on a mutant that also overproduces cysteine. However, the wildtype strains cannot produce methionine even in the presence of cysteine due to strict feedback regulation of methionine (Kumar et al., in press). Increased production of methionine is possible only with regulatory mutants. Therefore, it appears that a high methionine

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yielding strain would require not only feedback insensitive aspartate kinase and homoserinedehydrogenase, but also feedback insensitive serineacetyltransferase. In such a mutant, not only reverse trans-sulfuration will stop but also excess of cysteine will channel into methionine biosynthesis (trans-sulfuration) that will ultimately result in overproduction of methionine.

8. Media composition and culture conditions Success of an industrial fermentation depends greatly on careful selection of the nutrient medium (Chisti and Moo-Young, 1999). Media must contain all the requisite components in appropriate concentrations. Corynebacterium sp. requires biotin supplementation for growth. Media composition has a profound influence on microbial physiology and the ability to maximally produce a product is often associated with particular physiological forms (Ward, 1989). Microorganisms change their consortium of enzymes in response to the growth environment and, therefore, the media must be carefully designed to favor product formation. Banik and Majumdar (1974), Ghosh and Banerjee (1986) and Roy et al. (1989) have used empirical methods of media optimization and Sharma and Gomes (2001) used statistical method of media optimization for methionine production. Carbon sources, nitrogen sources and their ratio in fermentation media play a significant role in the production of particular metabolites (Chisti and Moo-Young, 1999). Various carbon sources have been reported for the production of l-methionine by various strains. Pham et al. (1992) used sugarcane juice, molasses, banana, cassava, and coconut water as carbon sources for methionine production. Glucose is the most widely used carbon source (Kase and Nakayama, 1974; Chattopadhyay et al., 1995a,b), but Banik and Majumdar (1974) reported maltose as the best carbon source for the production of methionine. Several workers have used methanol (Morinaga et al., 1982a,b; Tani et al., 1988) and n-alkanes (Ghosh and Banerjee, 1986) as main carbon sources. Various organic and inorganic nitrogen sources including urea, ammonium nitrate, ammonium sulfate, ammonium dihydrogen phosphate, ammonium chloride, sodium nitrate, ammonium acetate, ammonium tartarate, ammonium citrate, and ammonium oxalate have been used (Banik and Majumdar, 1975; Kase and Nakayama, 1975a,b,c; Yamada et al., 1982; Ghosh and Banerjee, 1986; Tani et al., 1988). Mondal et al. (1994) observed the effect of different nitrogen sources and different levels of biotin on methionine production and reported best methionine production at 60 mM ammonium nitrate and 5 Ag/l biotin. Although several researchers have used yeast extract as a N-source for methionine production, use of organic nitrogen sources is not advisable because they typically contain many amino acids (including methionine) and if a microorganism is provided with methionine in the medium it will not produce this amino acid. In addition to carbon and nitrogen sources, minerals and metal ions play a vital role in fermentation as metal ions are cofactors for various enzymes. The effect of various trace elements on methionine fermentation has been assessed (Banik and Majumdar, 1975; Morinaga et al., 1982a,b). Roy et al. (1989) reported the effects of sulfur compounds,


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metal ions and vitamins on growth and methionine production by Bacillus megaterium. They reported maximum methionine production using sodium sulfate as sulfur source, Fe2+, Mn2+ as metal ions and cyanocobalamine as vitamin. l-methionine-rich mutants of methylotrophic yeast Candida boidinii ICCF26 have high sulfur requirement and may be used as a single-cell protein (Avram et al., 1991). Kase and Nakayama (1974) have reported the effects of various amino acids on the production of O-acetyl-l-homoserine, an intermediate compound of metabolic pathway of methionine biosynthesis. Kase and Nakayama (1975a,b,c) reported media containing 10% glucose, 2% (NH4)2SO4, 0.05% K2HPO4, 0.05% KH2PO4, 0.1% MgSO4d 7H2O, 0.001% FeSO4d 7H2O, 0.001% MnSO4d 4H2O, 100 Ag/l biotin and 2% CaCO3 for methionine production. Banik and Majumdar (1975) reported 4.5 g/l methionine yield in media containing 5% maltose, 0.8% ammonium nitrate, 0.1% K2HPO4, 0.03% MgSO4d 7H2O, 1 mg/l Na2MoO4d 2H2O, 5 mg/l FeSO4d 7H2O, and 1 mg/l biotin at 7.0 pH. Tani et al. (1988) reported production media containing 1.5% methanol, 0.8% (NH4)2SO4, 0.1% KH2PO4, 0.2% MgSO4d 7H2O, 0.2% ZnSO4d 7H2O, 200 Ag/l thiamine hydrochloride and 2 Ag/l biotin at 5.5 pH for the production of methionine using methylotrophic yeast C. biodinii. Ghosh and Banerjee (1986) studied the effect of substances that affect the permeability of cells (e.g. penicillin, Tween 80, EDTA) and reported that these compounds did not increase methionine yield. Effect of oxygen on amino acid fermentation has been reported (Akashui et al., 1979; Hillinger and Hanel, 1981). Sharma and Gomes (2001) reported 40% dissolve oxygen as being optimal for the production of methionine using C. lilium NTE 99.

9. Recovery of methionine from fermented broth An inexpensive downstream recovery process that is capable of achieving the requisite recovery yield and purity, is essential for producing any metabolite (Chisti, 1998). Various levels of downstream processing are required for the existing amino acid fermentations (Hermann, 2003). The general approach to designing an efficient recovery scheme for bioproducts has been elucidated by Chisti (1998). The production scheme must accommodate the various regulatory requirements and consider the end use application of the product. Purification of amino acids relies on their physico-chemical properties, particularly solubility and isoelectric point. As the first step of the downstream recovery process, the cells are separated from the fermentation broth by either centrifugation or filtration. The cell-free broth is then passed through activated charcoal columns for decolorization. Methionine (isoelectric pH of 5.74) can be recovered from the clarified broth by adjusting the pH to 5.0 with sulfuric acid to convert the amino acid to its cationic form and passing the broth through a bed of Amberlite IR-120 (H+) ion exchange resin at a controlled flow rate. The process is repeated until all the methionine is adsorbed. Afterwards, the column is washed with deionized water and eluted with 1 M NH4OH to recover the methionine. Crystalline methionine can be obtained by concentrating under vacuum, treating with absolute alcohol and drying overnight at 4 8C (Banik and Majumdar, 1974; Roy et al., 1989).

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10. Concluding remarks Methionine is much in demand because of its increasing applications. Therefore, an economic process is required for producing methionine without the use of hazardous substances. The production requirements can be fulfilled potentially by fermentation processes but no such processes exist currently for commercial use. This is possibly due to insufficient knowledge of the severe feedback regulation of methionine biosynthesis. Classical methods of isolating high yielding strains by general mutagenesis have not been successful in providing an overproducer of methionine. A possible reason for this is that the assimilation of sulfur in the methionine biosynthesis has not been considered adequately. Direct sulfhydrylation, trans-sulfuration and reverse-trans-sulfuration processes play a significant role in methionine biosynthesis and its excretion from the cell. In view of the advances in molecular biology and genetics, information is now available for modifying the methionine biosynthetic pathway to relieve feedback inhibition. The undesirable feedback regulation because of lysine and threonine can be overcome by isolating a dual-auxotroph of lysine and threonine. Potentially, a high yield of methionine may be attained by lysine and threonine dual-auxotrophic mutant if it has deregulated enzyme systems that are insensitive to feedback inhibition or repression by methionine and cysteine.

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