production of l-methionine by submerged fermentation: a review

Enzyme and Microbial Technology 37 (2005) 3–18

Review

Production ofl-methionine by submerged fermentation: A review

James Gomes∗, Dharmendra KumarDepartment of Biochemical Engineering and Biotechnology, Indian Institute of Technology, New Delhi 110016, India

Received 24 June 2004; accepted 1 February 2005

Abstract

Production of methionine by various microorganisms is reviewed. The prospects of methionine production by submerged cultivation incomparison to other existing methods are examined. The metabolic regulation of methionine synthesis in several microorganisms in relationto difficulties of obtaining high-yield strains is presented in detail. An exhaustive survey of microorganisms that have been used to producemethionine, media compositions and analytical methods has been recorded. Details of reactor studies and metabolic flux analysis studies formethionine production have also been included.© 2005 Published by Elsevier Inc.

Keywords: l-Methionine; Submerged fermentation; Auxotrophic mutants; Regulatory mutants; Reactor studies; Metabolic flux analysis

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.1. History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.2. Clinical importance of methionine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.3. Industrial importance of methionine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2. Methods of methionine production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.1. Chemical synthesis of methionine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2. Enzymatic methods for methionine synthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3. Production of methionine by submerged cultivation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3. Methionine biosynthesis in microorganisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1. Metabolic regulation and control of methionine biosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Production of methionine by auxotrophic and regulatory mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95. Media composition and culture conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6. Analytical methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117. Process development and reactor studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
7.1. Production of methionine in batch, fed-batch and continuous reactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127.2. Modelling, simulation and control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

8. Metabolic flux analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139. Recovery of methionine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

10. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

∗ Corresponding author. Tel.: +91 11 2659 1013; fax: +91 11 2658 2282.E-mail address:[email protected] (J. Gomes).

0141-0229/$ – see front matter © 2005 Published by Elsevier Inc.doi:10.1016/j.enzmictec.2005.02.008

4 J. Gomes, D. Kumar / Enzyme and Microbial Technology 37 (2005) 3–18

1. Introduction

1.1. History

Methionine, an essential amino acid, was first isolatedfrom protein by Mueller[1] in 1922 and its structure iden-tified as the�-methylthiol derivative of�-amino-n-butyricacid by Barger and Coyne[2] in 1928. Its function in trans-methylation was inferred indirectly from evidence obtainedin studies in animal nutrition by Vigneaud et al.[3] in1939, who observed that homocysteine could replace me-thionine in animal diets if they were also supplemented withcholine or betaine. More than a decade later, in 1953, Cantoni[4] confirmed the transmethylation function of methionineby identifying S-adenosylmethionine (SAM) as a primarymethylating agent. In 1958, Tabor et al.[5] established itsrole as a precursor of polyamines. The elucidation of an-other important function of methionine, its participation intranslation of m-RNA by incorporating itself as the universalN-terminal amino acid in a growing peptide, resulted fromthe research of Adams and Capecchi[6] in 1966 and Clarkand Marcker[7] in 1968. A more detailed account of the his-tory of methionine has been given by Greenstein et al.[8] andFlavin [9].

The discovery of microbial production of glutamic acid[10] triggered the search for other amino acid producingm anyr to de-v ub-m eb ef incta heticm uldb h asl pro-d -p df

onh ismse uceds de-m crosst ly.M anda o thecm .P s, eacs efforta sim-p ow-e sh .I cess

biotechnology, the viability of commercial production ofl-methionine needs to be re-examined. In later sections, thestatus of methionine production by various methods andkey issues related to submerged fermentation of methionineare discussed.

1.2. Clinical importance of methionine

Methionine is required in small amounts in the diet ofhumans and other mammals for normal growth and bodyfunctions. In humans, some rare hereditary diseases like cys-tathioninuria and homocystinuria are caused by defectivemetabolism of methionine. Both cystathioninuria and homo-cystinuria are inherited as autosomal recessive genetic traits.Patients suffering from these diseases may exhibit one ormore symptoms such as, mental retardation, seizures, throm-bocytopenia, clubfoot, skeletal abnormalities, lens disloca-tion, and hearing defects[30–33].

Dietary deficiency of methionine has been linked to suchailments as toxemia, childhood rheumatic fever, muscleparalysis, hair loss, depression, schizophrenia, Parkinson’sdisease, liver deterioration, and impaired growth[34–36].Methionine functions as building block of proteins and asa source of polyamines. It is a precursor of SAM, whichfurnishes labile methyl groups and sulfur to over 100 reac-tions. Many of these reactions are involved in the synthe-s ns.I ationit anda cem thee hu-m d byt on-i leveli erio-r tomsi g-g tomso r-t fo-l pedi em-p inB d tot omep

anm pHa ticala top8i avea ofd

icroorganisms[11–15]. Since then, there have been meports about the efforts made by several researcherselop high-yielding strains for producing methionine by serged fermentation[16–23]. The production of methioniny submerged cultivation produces the biologically activl-

orm of the amino acid. This method would have distdvantages over the existing synthetic and semi-syntethods provided a sufficiently high concentration coe obtained. Although several other amino acids suc

ysine, threonine, isoleucine and histidine, are beinguced successfully by fermentation[24–29], there are no reorts about the production ofl-methionine by submerge

ermentation.The production ofl-methionine by submerged cultivati

as not been adopted commercially because microorganither mutated or genetically engineered, have not produfficiently high concentrations of methionine. The bulkand for methionine, about 350,000 tonnes annually a

he globe, is met by thedl-methionine produced chemicalost of the methionine demand comes from the poultrynimal feed industry and the remaining demand is due tlinical and therapeutic use ofl-methionine. Thel-form ofethionine is obtained by racemisation of thedl-methioninerocess development takes place through several stagetage requiring a substantial investment of researchnd funding and may not be desirable considering theler and economical chemical method of production. Hver, the use of purel-methionine in clinical applicationas increased, creating a niche market forl-methionine

n view of the recent developments in the area of pro

,

h

is of compounds that are vital to cell or organ functiot acts as a lipotropic agent and prevents fat accumuln the liver by increasing lecithin production[37,38]. Me-hionine is essential for the absorption, transportationvailability of selenium and zinc in cellular functions. Sinethionine is an excellent chelating agent, it helps in

xcretion of cadmium and mercury from the body. Inans, methionine is used to produce creatinine require

he brain. People with AIDS have a low level of methine. Some researchers suggest that this low methioninen AIDS patients may explain some aspects of the detation that occurs in the nervous system causing sympncluding dementia[39]. Also, preliminary trials have suested that methionine may help to treat some sympf Parkinson’s disease[39]. Methionine plays an impo

ant role in regulating the physiological availability ofate. When methionine level is low, folate becomes trapn the liver as 5-methyl-tetrahydrofolate, causing a torary folic acid deficiency. This is also seen in Vitam12 deficiencies and may be an important factor linke

he cause of allergy in many patients and anemia in satients.

The l-form of methionine is used extensively in humedicine for a variety of therapeutic purposes, includingnd electrolyte balancing, parental nutrition, pharmaceudjuvant, and other applications. In fact, it is one of the00 drugs in human medicine[40]. Thed-form of methion-

ne is not well utilized in humans and individuals may hllergic reactions tod-methionine or the racemic mixturel-methionine[41].

J. Gomes, D. Kumar / Enzyme and Microbial Technology 37 (2005) 3–18 5

1.3. Industrial importance of methionine

The use of methionine in the poultry and livestock indus-tries has an indirect influence on humans. Methionine is thefirst limiting amino acid in poultry diets. The use of syntheticmethionine in animal feed began in 1950s with the simplifi-cation of poultry feed by adding synthetic constituents[42].Methionine deficiency has become a problem in recent yearsdue to the increasing use of low cost soybean protein andbird genetic selection for increase broiler growth or egg lay-ing ability. Methionine is metabolically linked with cysteineand choline and is necessary for producing keratins used infeather growth. In livestock, for the optimum performanceof the animal, its diet must contain adequate quantities of allnutrients, including amino acids. A shortage of methionine,the limiting amino acid, reduces feed efficiency, constrainsgrowth and in extreme cases results in nutritional deficiency.Supplementation with isolated amino acids increases feedconversion efficiency, thus lowering feed costs per unit gainof the animal. Amino acids are also used in livestock health-care. Methionine is used as a urine acidifier because excretionof its sulfate anion lowers urine pH. The sulfate anion alsodisplaces phosphate from uroliths and thereby assists in dis-solving and preventing kidney stones and bladder stones[43].

2

2

ce ad arem e thes withv ce ofa ro-g diatesap is re-a xideim com-m inoa ird,u . Theu imalfHh alsor bi-o ea

2

en-z

methionine using amino acylases[50–56]. A continuous res-olution technique using a column of immobilized fungalaminoacylases for resolving a mixture ofdl-methionine wasdeveloped for commercial use by Tosa et al.[52]. Mori-naga et al.[53] reportedl-methionine production fromdl-homocysteine and methanol using resting cells of ethionineresistant mutant ofPseudomonasFM 518. Morinaga et al.[54] have also used resting cells of ribulose-monophosphatepathway-type methylotroph strain OM 33 to catalyse the for-mation of methionine fromdl-homocysteine and methanol.They obtained 26 mM ofl-methionine from 30 mM ofdl-homocysteine and 0.8 mM methanol. Soda[55] reported bio-conversion ofN-carbamylmethionine intol-methionine byculturingBacillussp. 266 andVibrio sp. 256, and obtained34.4% and 25.5% methionine yield, respectively. Yamashiroet al. [56] reported bioconversion ofdl-5-substituted hy-dantoins intol-form of amino acids including methionineby Bacillus brevisand its mutant. Voelkel and Wagner[57]reported bioconversion ofdl-5-metylthioethylhydantoin byresting cells ofArthrobactorsp. DSM 7330 andAspergillussp. Wagner et al.[58] reported bioconversion ofdl-5-(2-methylthioethyl)-hydantoin into methionine with 90%yield by resting cells of the mutant strain DSM 9771 us-ing fed-batch technique. Using resting cells the optimalbiotransformation parameters were pH 7.5 and tempera-ture 37◦C. No inhibition of the enzymatic system couldb ubil-iu haaT ono m-b no -sa ldw

ofd teinh -a hy-d re ofa

2c

cingm ackr it.N cea rea-s rym vel-o na-l tants

. Methods of methionine production

.1. Chemical synthesis of methionine

Chemical routes for the synthesis of methionine produl-mixture of the amino acid. Currently, all these routesodifications of the Strecker synthesis, and they shar

ame raw materials: acrolein, methyl mercaptan, alongarious sources of ammonia and cyanide, in the presencatalyst[44–46]. Another method uses propylene, hyd

en sulfide, methane and ammonia, to make the intermecrolein, methylthiol, and hydrocyanic acid[47]. In a recentlyatented process, 3-methyl-mercaptopropionaldehydected with ammonia, hydrogen cyanide, and carbon dio

n the presence of water in three reaction steps[48]. Thedl-ethionine hydroxy analogs are also produced and usedercially. The hydroxy analogs are converted to the amcid form by transamination in the liver of the animal or bsing non-essential amino acids such as glutamic acidse of methionine and other synthetic constituents in an

eed has simplified feed preparation and formulation[49].owever, the synthesis ofdl-methionine anddl-methionineydroxy analogs for this purpose by chemical methodsesults in environmental pollution. Therefore, alternativelogical methods for producingl-methionine would be morcceptable.

.2. Enzymatic methods for methionine synthesis

Biologically activel-methionine has been producedymatically by the stereospecific cleavage ofN-acyl-dl-

e detected up to the maximum concentration of solty of dl-5-(2-methylthioethyl)-hydantoin (30 g l−1). These of cystathionine-�-synthase in the production of alpmino acids has also been reported in the literature[59].okuyama and Hatano[60] reported continuous productif l-methionine in a bioreactor with 99% yield using recoinantEscherichia coliMM294 (DE3) with over expressiof the gene forN-acylamino acid racemase fromAmycolatopissp. TS-1-60 donor strain. Imi and Shiozaki[61] reportedprocess to producedl-methionine with an improved yiehile reducing the load of wastewater disposal.Other methods include the enzymatic resolution

l-methionine produced by chemical synthesis and proydrolysis. While the chemical processes[62–65] use hazrdous raw materials, methionine production by proteinrolysis requires several stages for separating the mixtumino acids.

.3. Production of methionine by submergedultivation

The most serious problem encountered in produethionine by submerged cultivation is the strict feedb

egulation exercised by microorganisms in producingormally wild type microorganisms do not overprodumino acids, in particular methionine because of thison [66,67]. Mutants with genetically altered regulatoechanism and exhibiting high yields need to be deped for the production of methionine. Methionine a

ogues have widely been used to isolate such mu


because they effectively function as true feed back inhibitorswithout participating in other useful functions in the cells[68]. Mutants resistant to methionine analogues have en-zymes insensitive to feed back inhibition and repressionand such mutants overproduce methionine in culture broth[12,18,19,22]. Although attempts have been made by sev-eral researchers to produce methionine by using microorgan-isms[20], no process development research has been reportedtill date.

3. Methionine biosynthesis in microorganisms

About sixteen different species of microorganisms havebeen reported to produce methionine (Table 1). Among these,Corynebacteriumor Brevibacteriumspecies have much sim-pler regulatory mechanisms. InFig. 1, the pathway forCory-nebacteriumsp. and inFig. 2, the pathwayE. coli is presented[69]. The evolutionary reasons for this simplification may bespeculated upon but remains uncertain. Since the pathway

Table 1A list of microorganisms producing methionine

S. no. Microorganism Genetic marker Methionine yield(mg ml−1)

Method of methionineanalysis

1. Arthrobactersp. DSM9771[58] – 23.00a HPLC

2. Bacillus megaterium[22,142]B6US-215 – 4.50 PCB71 – 0.072 PC, MB

3. Brevibacterium heali[18,19]BhLT 27 Lys−, Thr− 25.5 –BrEthR50

2 EthR 13.0 PC, MB

4. Candida boidinii[105]2201 – 6.2 mg g−1

MBE500-78 EthR 16.02 mg g−1b

AKU 4618 – 4.54 mg g−1

Candidasp. 25 A – 6.12 mg g−1

5thR

thR

thR

thR, SLMR

thR, TriathR, TriathR TriathR, MethR, SL

67

89

1

111111

ANs

. C. glutamicum[12]ER 107 Thr−, EER 108 Thr−, EER 109 Thr−, EESLMR 724 Thr−, EESLR 146 EthR, SLETz R 754 Thr−, EETz R 606 Thr−, EETzFR 619 Thr−, EETz MR 510 Thr−, EESLFR 736 Thr−, E

. C. lilium M128 [70] EthR, Norleucin

. E. coli [68,108,109]K12 5BUR

K12 AHVR, EthR, 5BJM109-TNI EthR, Norleucin

. Hansenula polymorphaDL1 [105] –

. Kluyvermyces lactisIPU126[141] EthR

0. Methylomonassp.[23]PC, MBOM33 EthR

OE120 EthR

1. Micrococcus glutamicusX1 [16] Biotin−2. Pichia pastorisIFO 0948[105] –3. Pseudomonassp. FE-244[21] EthR

4. Pseudomonas putidaVKPMV-4167 [110] EthR

5. Serratia marcescensvar.kiliensis[107] –6. Torulopsis glabrataIFO 005[105] –

bbreviations for methionine analysis method: COL, colorimetry; MB, micro. 8042); PC, paper chromatography; SP, spectrophotometry; AAA, aminoelenomethionine; MetS, methionine sulphoxide; 5BU, 5-bromouracil; AHV,�-ama Hydantoin bioconversion.b Productivity in optimized medium.c Productivity when cysteine was added in medium.d n-Alkane as substrate.

0.100; 0.200c

COL,PC,MB

0.100; 0.150c

–; 0.500c

MR MetHxR 2.000.550

zoleR, TFMR 0.754zoleR, 0.610

zoleR, TFMR 0.950tHxR, TriazoleR 0.850MR, TFMR 0.100

eR, MetSoR 1.98 SP

1.00 –UR 2.00 PCeR 0.91 AAA, MB

5.00 mg g−1 MB14.20 –

0.070.42

2.00 PC5.60 mg g−1 MB0.80 MB3.5 –0.78d PC5.04 mg g−1 MB

obiological assay (all reported assays usedLeuconostoc mesenteroidesATCCacid analyser. Abbreviations markers: MetHx, methionine hydroxamate; SLM,

ino-�-valeric acid.


Fig. 1. Regulation of methionine biosynthesis inCorynebacteriumor Brevibacteriumsp.[63]: (—) inhibition; (- - -) repression.

for methionine synthesis is so strictly regulated, mutants withderegulated pathways need to be isolated. Many such organ-isms have been isolated (Table 1), indicating in a sense itsindustrial importance. Likewise, efforts have also being madeto produce methionine by submerged fermentation[70].

Methionine, lysine, threonine and isoleucine are the mem-bers of aspartate family of amino acids. The pathway forthe biosynthesis of amino acids of this family has been elu-cidated from the cumulative work of several researchers[71–78]. Wijesundera and Woods[79] and Rowbury and

isscheri
Fig. 2. Regulation of methionine biosynthesE chia coli[63]: (—) inhibition; (- - -) repression.


Woods[66,73]reported the complete metabolic pathway formethionine biosynthesis inE. coli. Flavin et al.[80] have re-ported methionine biosynthesis inSalmonella typhimurium.Methionine biosynthesis and its regulation inCorynebac-terium were reported by Kase and Nakayama[12,13]. Thepathway of methionine biosynthesis has many common fea-tures in most organisms, even though some species utilizedifferent biosynthetic routes but it is synthesised de-novoby most bacteria and fungi. According to this pathway as-partate is converted to 4-phospho-l-aspartate by aspartatekinase, which is oxidised by aspartaldehyde dehydrogenaseto form aspartate semialdehyde. Aspartate semialdehyde isoxidised by homoserine dehydrogenase to form homoser-ine. On the other hand, aspartate semialdehyde is convertedto dihydropicolinate by dihydropicolinate synthase, whichleads to the formation of lysine. From homoserine, twobranches arise, one leads to the formation of methionine whileother leads to the formation of threonine and subsequentlyisoleucine. Homoserine undergoes condensation with suc-cinyl CoA and produceO-succinylhomoserine inE. coli byhomoserineO-succinyltransferase. Most of the bacteria haveO-succinylhomoserine as an intermediate while most of thefungi and few bacteria likeBacillus and CorynebacteriumhaveO-acetylhomoserine instead ofO-succinylhomoserine[80]. Formation of methionine fromO-acetylhomoserinemay take place by two possible metabolic pathways. Accord-i thio-n i likeN e isd e( r-vw dO the-s en-z lea oninei ia byc -p nb iono mo-c to uta-m ases.R eb -i

3b

siso sev-e thefi s of

aspartate family which catalyses the phosphorylation of as-partate. InE. coli, there are three distinct aspartate kinases.Aspartate kinase I is inhibited by threonine and its synthesisis repressed by threonine and isoleucine. Aspartate kinase IIis inhibited and repressed by methionine and aspartate ki-nase III is inhibited and repressed by lysine. Therefore, anexcess of one product does not shut down the entire pathway[67]. The reduction of aspartate-semialdehyde to homoser-ine is catalysed by two distinct homoserine dehydrogenases(EC 1.1.1.3). Synthesis of homoserine dehydrogenase I isrepressed by threonine and isoleucine and its activity is in-hibited by threonine, whereas homoserine dehydrogenase IIis repressed by methionine. In addition,E. coli is able to ad-just the presence of unbalanced mixture of the products of thepathway. If it encounters with an excess of methionine levelbut low levels of lysine, threonine and isoleucine then regulat-ing aspartate kinase alone will not lead to the proper ratio ofmethionine to the other three amino acids. Thus, each aminoacid usually controls the first enzyme of its own particularbranch. On the other hand,BrevibacteriumandCorynebac-teriumsp.were found to have much simpler regulation. Thesemicroorganisms have only one aspartate kinase inhibited bylysine and threonine in a concerted manner. Therefore, ef-ficient feedback inhibition of aspartate kinase requires bothlysine and threonine (Fig. 2). When lysine and threonine arepresent simultaneously at 1 mM, they inhibit aspartate kinaseb M, in-h

hesisi rts ap olvedi u-l syn-t M ori thec ssese ipa-t va-t t inte ineb atm e ofB pe-c sc thefi syn-t . InE utedt sop ing inv -ti me-t d

ng to first one, homocysteine is synthesized from cystaine as in case of some eccentric bacteria and few fungeurospora crassa. In the second one, acetyl homoserinirectly converted to homocysteine byO-acetylhomoserinthiol)-lyase[81]. Morinaga et al.[82] made similar obseations in facultative methylotroph,PseudomonasFM 518hich showed the activity of both�-cystathioninase an-acetylhomoserine sulfhydrilase. Cystathionine is synised fromO-succinyl homoserine and cysteine by theyme cystathionine�-synthase[80]. This step is reversibnd requires pyridoxal phosphate as a cofactor. Cystathi

s then cleaved to homocysteine, pyruvate and ammonystathionine-�-lyase (EC 4.4.1.8) (metC), a pyridoxal phoshate dependent enzyme[73]. Homocysteine on methylatioy homocysteineS-methyltransferase leads to the formatf methionine. The methylation of homocysteine by hoysteineS-methyltransferase may be Vitamin B12 dependenr independent. But 5-methyltetrahydrofolate (a polyglate derivative) acts as a methyl donor in both the cecently, Rukert et al.[83] elucidated the pathway for thiosynthesis ofl-methionine inCorynebacterium glutam

cumusing genome sequences (Fig. 3).

.1. Metabolic regulation and control of methionineiosynthesis

Metabolic regulation and its control for the biosynthef amino acids of aspartate family have been studied byral workers[14,61]. Aspartate kinase (EC 2.7.2.4) isrst enzyme of the metabolic pathway of the amino acid

y 94%, whereas each amino acid, present alone at 1 mibits aspartate kinase marginally by 12–20%[69].

Studies on regulatory aspects of methionine biosyntn C. glutamicumshows that exogenous methionine exeotent repression of the synthesis of some enzymes inv

n this pathway[11]. Mondal et al.[20] has recorded the regation of some of the enzymes involved in methionine biohesis. It has been reported in some organisms that SAts derivative rather than methionine itself, is likely to beo-repressor of the methionine biosynthesis. SAM reprenzymes involved in methionine biosynthesis. The partic

ion of SAM in repression together with inhibition obserion indicates that SAM is likely to be a kind of end produche methionine biosynthesis inCorynebacterium[13]. Syn-rgistic feedback inhibition of the first enzyme of methioniosynthesis is also found inBacilli. It has been reported thethionine and SAM inhibit homoserine acetyltransferasacillus polymyxaand each inhibitor binds to a different sific site distinct from the active site[84]. In Saccharomyceerevisiae, SAM inhibits homoserine acetyltransferase,rst enzyme of the metabolic pathway of methionine biohesis, individually rather than in concert with methionine. coli, the genes of the methionine regulon are distrib

hroughout the chromosome[85]. The regulatory functionf S-adenosylmethionine as co-repressor and of themetJ generoduct as aporepressor have been confirmed by studyitro expression of some of theE. coli methionine biosynhesis genes[86,87]. However, none of themetJ andmetKsolated regulatory mutants were fully decontrolled forhionine production[88,89]. Patte and Boy[71] have reporte


Fig. 3. Methionine biosynthesis inC. glutamicum[83]: [1] aspartate kinase; [2] aspartaldehyde dehydrogenase; [3] homoserine dehydrogenase; [4] homoserineO-acetyltransferase; [5] O-acetylhomoserine (thiol)-lyase (cystathionine�-synthase); [6] cystathionine�-lyase; [7] homoserineS-methyltransferase (VitaminB12 independent or metH-encoded, Vitamin B12 dependent); [8] homoserine kinase; [9] threonine synthase; [10] O-acetylhomoserine sulfhydrylase; [11] serinehydrooxymethyle transferase.

that inE. coli the enzyme aspartaldehyde dehydrogenase isof only one kind and is multi-valently repressed by lysine,threonine and methionine. Studies by Urbanowski et al.[90]have identified a regulatory locus, calledmetR, required forthe expression ofmetE andmetH genes. However,metE geneis shown to auto-regulate its own synthesis during in vitrostudies while metR protein stimulates its in vitro expression[91]. The metR gene is a trans-activator of the expressionof metE gene andmetR gene is under autogenous regulationand is repressed by metJ protein[92]. Two of the enzymesinvolved in the terminal steps of methionine biosynthesis arerepressed in a non co-ordinate manner by both Vitamin B12and methionine inE. coli [93]. Recently, Kalinowsky et al.[94] provided the architecture of the genome ofCorynebac-terium glutamicumand used it to reconstruct the metabolicpathways to produce amino acids of aspartate family.

4. Production of methionine by auxotrophic andregulatory mutants

Most of the amino acids are produced industrially by us-ing regulatory or auxotrophic regulatory mutants. These aux-

otrophic mutants are generally useful for the production of in-termediate metabolite of the straight chain pathway but theseare also useful for the production of branched pathway aminoacids such as lysine and methionine. As in case of methionineproduction using auxotrophic mutants (threonine auxotroph;lysine and threonine dual auxotroph) has two advantages (i)inhibitory effects are eliminated and (ii) dissipative carbonflux to branch pathways are cut off. Consequently, aspartatekinase and homoserine dehydrogenase are not inhibited bylysine and threonine, and this leads to higher production ofmethionine.

Regulatory mutants for the production of methionine canbe isolated using analogues of methionine. Analogues inhibitgrowth of the wild type strain in minimal media. Earlier, theprevailing view was that these analogues inhibit growth bygetting incorporated into proteins and thereby producing thenon-functional proteins. This may well occur in some in-stances, but in vast majority of the cases, the major cause ofinhibition appears to be that the analogues mimic the wayof amino acid which regulates its own production. Thus ana-logues may bind to the allosteric or the product site of theenzyme or may bind effectively to the repressor and resultingshut down of the pathway for the synthesis of that particular


amino acid. It inhibits growth of organism because of the star-vation of that amino acid. Therefore, amino acid analoguesact as pseudo-feedback inhibitors or repressors thereby in-hibiting or repressing the synthesis of the correspondingamino acid. Only mutants having resistance to analogues mayproduce corresponding amino acid in excess, which is even-tually excreted out of the cells to the broth. These mutantsare able to resist the analogues either because of an alterationin the structure of the enzyme or an alteration in the enzymeforming system. In the case of methionine, analogues such asethionine, seleno-methionine, norleucine and methionine hy-droxamate have been used to develop overproducing strainsin various microorganisms[12–14,18,19]. A list of methion-ine producing microorganisms is given inTable 1. Modes ofanalogue resistance are as follows: (1) mutation in an oper-ator, resulting in de-repression of biosynthetic enzymes; (2)mutation in a structural gene of a biosynthetic enzyme sub-jected to end product inhibition, resulting in loss of feed backcontrol; (3) mutation in structural gene of an amino acyltrans-fer RNA synthase, resulting in discrimination between aminoacid and the analogue; (4) mutation in the structural gene ofa permease for the amino acid, resulting in reduced ability totake up the analogue.

Auxotrophic mutants with resistance to analogues arewidely used for commercial production of amino acids. Itwas possible to obtain higher yields of lysine by simply cut-t egu-l e7w[ d7 eenr erm ula-t

byr nt ofy antm -r t,C t,Ct y-lt

mA sm rry-i lastso ibi-o -p hiesam utanto

nine in a medium containing 10% glucose[12]. Ghosh andBanerjee[107] reported that a hydrocarbon utilizingSerratiamarcescensvar. kiliensisproduced 1.68 g l−1 glutamic acidand 0.78 g l−1 methionine in optimised culture conditions ina synthetic medium with hydrocarbon as the sole carbonsource. A mutant ofE. coli K-12, resistant to methionineanalogues (norleucine, ethionine and�-methylmethionine)and threonine analogue,�-amino-�-hydroxy valeric acid,produced 2 mg ml−1 of both methionine as well as threo-nine [68]. Ethionine and 5-bromouracil resistant strain ofE. coli K-12 was reported to produce 1 mg ml−1 of bothmethionine as well as threonine[108]. Methionine ana-logue resistant mutant strain TN1 ofE. coli JM109 pro-duces 910 mg l−1 l-methionine[109]. Odessa[110] reported3.5 g l−1 l-methionine production by adl-ethionine resistantmutant ofPseudomonas putidaVKPM V-4167.

Genencor[111] has reported that methionine productionby microorganisms can be enhanced by modifying the path-way of methionine biosynthesis in a microorganism whichinvolves: (a) transforming or transducing the host with ahomoserine activating enzymes (e.g. homoserine kinase),homoserine acetyltransferase or homoserine succinyltrans-ferase gene fragment and a sulfur incorporating enzyme (e.g.O-succinylhomoserine-(thiol)-lyase orO-acetylhomoserine-(thiol)-lyase) gene fragment; (b) then recovering the trans-formants; and adding an exogenousS-source to the cells formo seda

re-p ine.A ichw ncedt midtc e ort rtedi thea

5

osi-t n allt r theg ediao ha om-p oor-g iatedw lc er toa forem iron-m dar

ing off the branches leading to other amino acids. Ratory mutants ofCorynebacteriumare reported to produc9 g l−1 lysine[95], 54 g l−1 lysine[96], 48 g l−1 lysine[97]hile auxotrophic regulatory mutants produce 70 g l−1 lysine

98] and 76 g l−1 lysine[99]. Threonine production of 72 an6.5 g l−1 by auxotrophic regulatory mutants has also beported[100,101]. Mondal et al.[18] reported that ethioninesistant mutants ofBrevibacteriumhealiproduced 13 mg l−1

ethionine, while lysine and threonine auxotrophic regory mutants ofB. healiproduced 25.5 g l−1 methionine[19].

Expanding the intracellular pool of free methionineegulatory mutants can increase the methionine conteeast.l-Methionine overproduction by ethionine resistutants has been reported forS. cerevisiae[102], Saccha

omyces lipolytica[103] and ann-paraffin utilising yeasandida petrophilum[104] while a methylotrophic yeasandida boidiniino. 2201 produced 6.2 mg g−1 DCW of

otal methionine[105]. Ethionine resistant mutant of methotrophic bacteria is reported to produce 420�g ml−1 me-hionine under optimised conditions[23].

Ethionine resistant mutants ofSaccharomyces uvaruTCC 26602 were found to overproduce exogenoul-ethionine. Adl-ethionine resistant mutant ER 108, ca

ng a mutation to chloramphenicol resistance, and protopbtained from it were fused with protoplasts from anttic sensitiveS. cerevisiaeX2928 carrying six auxotrohies. The resulting fusants maintained four auxotropnd were capable of overproducingl-methionine[106]. Aulti-analogue resistant and threonine auxotrophic mf C. glutamicumESLMR 724 produced 2 mg ml−1 methio-

ethionine production, host may be aCorynebacteriumsp.rBrevibacteriumsp. Use of methionine rich yeast to be us single cell protein is also reported in the literature[112].

Use of modern molecular biology techniques is alsoorted in the literature to obtain a better yield of methionpoly-methionine DNA fragment containing plasmid whas transformed into a parent yeast cell CB89, enha

he methionine production in comparison to simple plasransfer[112], while a DNA inversion gene containingE.oli shows hyper secretion of phenylalanine, methioninyrosine[113]. Amino acid overproduction has been repon S. cerevisiaemutants where degradative pathways formino acids are blocked[114].

. Media composition and culture conditions

An industrial fermentation greatly depends on compion of medium. Media used in fermentation must contaihe components in appropriate concentration required forowth and product formation that can be adjusted by mptimisation.Corynebacteriumsp. requires biotin for growtnd it must be a constituent of any medium. Medium cosition has a profound effect on the physiology of micranisms and maximum product formation is often associth particular physiological forms[115]. Since bacteriaells can change patterns of enzyme synthesis, in orddapt themselves to their specific environment, thereedia must be designed to provide a favourable envent for particular product formation. Banik and Majum


[17], Ghosh and Banerjee[107] and Roy et al.[22] haveused empirical media optimisation for methionine produc-tion. Sharma and Gomes[116] used statistical methods formedia optimisation.

Carbon sources, nitrogen sources and their ratio in fer-mentation media play a very significant role in the produc-tion of particular metabolite. Various carbon sources havebeen reported for the production ofl-methionine by var-ious strains. Of these carbon sources, glucose is the mostwidely used[11,68,108]although Banik and Majumdar[17]had reported maltose as a best carbon source for the pro-duction of methionine. Several workers have used methanol[21,105]andn-alkanes[107]as a main carbon source for me-thionine production. Pham et al.[117] used sugarcane juice,molasses, banana, cassava, and coconut water as a carbonsource for the production of methionine by fermentation.Various inorganic nitrogen sources including urea, ammo-nium nitrate, ammonium sulphate, ammonium dihydrogenphosphate, ammonium chloride, sodium nitrate, ammoniumacetate, ammonium tartarate, ammonium citrate and ammo-nium oxalate have been reported for methionine production[12–14,17,23,10,107]. Mondal et al.[19] have observed theeffect of different nitrogen sources and different levels of bi-otin on methionine production and reported best methionineproduction at 60 mM ammonium nitrate and 5�g l−1 biotin.Although, several researchers have used yeast extract as a ni-t ablet moso antsb eralso them rkersh ninef ul-p me-tm e ass al-t l-t urr

usa ni nineb ac00m4 on-t0F l.[ nol,00 d

2�g l−1 biotin at pH 5.5 for the production of methionineusing methylotrophic yeastCandida biodinii. Ghosh andBanerjee[107] studied the effect of substances that affect thepermeability of cells, like penicillin, tween 80 and EDTA andreported that these compounds do not increase methionineyield. Effect of oxygen on amino acid fermentation has beenreported in the literature[119,120]. Sharma and Gomes[116]reported 40% dissolve oxygen optimal for the production ofmethionine usingCorynebacterium liliumNTE 99. Kumaret al.[70] have reported that cysteine inhibits the growth ofwild type strains whereas it increases methionine productionwhen added to the production media of the mutant strain.

6. Analytical methods

A number of methods have been developed for theestimation of methionine in culture broth. These methodsinclude chemical analysis[121], paper chromatography[122], colorimetric analysis[8], gas liquid chromatography[123] gas chromatography–mass spectrometry[124], ionexchange infrared spectroscopy[125] and high performanceliquid chromatography[126–128].

Fink et al. [129] have reported a paper chromatogra-phy method using a solvent system comprisingn-butanol,acetic acid and water in a 2:1:1 ratio. A reaction that hasb n thec fn hust witht hro-m d ofe o-r rami ota fluo-rc grayc ht,wa inga teri a-t velyg

edc ofm estt bef us-s edt uss ithf nal1 di lour

rogen source for methionine production but it is not adviso use organic nitrogen sources because these containf the amino acids and increase the likelihood of reverteing formed. Besides carbon and nitrogen sources, minr metal ions play a vital role in fermentation, as most ofetal ions are cofactor for various enzymes. Several woave reported the effect of various minerals on methio

ermentation. Roy et al.[22] have reported the effects of shur compounds, metal ions and vitamins on growth and

hionine production byBacillus megaterium.They reportedaximum methionine production using sodium sulphat

ulphur source, Fe2+, Mn2+ as metal ions and cyanocobamine as vitamin.l-Methionine rich mutants of methyrophic yeastCandida boidiniiICCF26 have higher sulphequirement and may be used as a single cell protein[118].

Kase and Nakayama[11] have reported effect of variomino acids on production ofO-acetyl-l-homoserine, a

ntermediate compound of metabolic pathway of methioiosynthesis. Kase and Nakayama[12] reported mediontaining 10% glucose, 2% (NH4)2SO4, 0.05% K2HPO4,.05% KH2PO4, 0.1% MgSO4·7H2O, 0.001% FeSO4·7H2O,.001% MnSO4.4H2O, 100�g l−1 biotin and 2% CaCO3 forethionine production. Banik and Majumdar[17] reported.5 g l−1 methionine yield in culture broth using media c

aining 5% maltose, 0.8% ammonium nitrate, 0.1% K2HPO4,.03% MgSO4·7H2O, 1 mg l−1 Na2MoO4·2H2O, 5 mg l−1

eSO4·7H2O, and 1 mg l−1 biotin at pH 7.0. Tani et a105] reported production media containing 1.5% metha.8% (NH4)2SO4, 0.1% KH2PO4, 0.2% MgSO4·7H2O,.2% ZnSO4.7H2O, 200�g l−1 thiamine hydrochloride an

teen described as specific for methionine is based oolour, which forms after the treatment with a solution o�-aphthylamine diazoniumchloride in hydrochloric acid. T

he pertinent amino acid mixture is separated on paperhe aid of a suitable solvent system and the resulting catogram dried and sprayed with a mixture composequal volumes of 0.1%�-naphthylamine in 10% hydrochlic acid and 0.5% sodium nitrite in water. The chromatogs dried for 5 min at 80◦C, after which the methionine spppears as an orange yellow hue that exhibits a dark redescence under ultra violet light. As little as 1�g methioninean be detected by this method. Tryptophan gives a blueolour with a yellowish fluorescence under ultra violet lighile the other common amino acids fail to react[8]. Kasend Nakayama[11] reported methionine determination ussolvent system comprisingn-butanol, acetic acid and wa

n a 4:1:2 ratio, andn-butanol, formic acid, methanol and wer in a 4:1:2:0.5 ratio. The latter was found to be relatiood for the separation of methionine.

In another method[8], methionine has been determinolorimetrically. Test sample (containing 0.1–1.0 mgethionine) is diluted to 5.0 ml with distilled water in t

ube. One milliliter of 5 N NaOH is added to each tuollowed by the addition of 0.1 ml of 10% sodium nitropride solution with through mixing. The mixture is allowo stand for 10 min. Then two milliliters of 3% aqueoolution of glycine is added to the reaction mixture wrequent shaking over a period of 10 min. After an additio0 min interval, 2 ml of concentratedortho-phosphoric aci

s added drop wise to the mixture with shaking. Co


development is allowed to proceed for 5 min and the colourintensity measured at 540 nm in a spectrometer. A blankcontaining distilled water and all other reagent serves asthe 100% transference standard. Results obtained with thetest samples are interpolated on a standard curve. Larry etal. [126] has reported a rapid, quantitative method by highperformance liquid chromatographic (HPLC) procedurefor the determination of methionine and cysteine. Jones etal. [130] reported a rapid and ultra sensitive fluorescencemethod for amino acid analysis usingO-pthaldialdehydeas derivatising agent.O-Pthaldialdehyde in the presence ofmercaptan reacts rapidly with primary amino acids to firmintensely fluorescent derivatives that are analysed with goodselectivity by HPLC. Herbert et al.[131] reported a methodfor the measurement of free amino acids in biological fluidsby HPLC usingO-pthalaldehyde-3-mercaptopropionic acidas derivatising agent. Multiple gradient systems were usedfor the analysis of free amino acids in physiological fluids byHPLC with fluorescence detection on two reverse phase C18columns. More than 22 amino acids were separated in lessthan 1 h on either 5�m Ultrasphere-ODS columns or 5�mResolve C18 columns by using a two solvent system[132].

7. Process development and reactor studies

7c

-d andN ins nt oft s ofb thio-

nine producing NTG mutants,C. lilium M128. More than2.3 g l−1 of methionine was obtained at the end of 48 h offermentation. These experiments were carried out in a 15 lreactor.

The only reports on methionine production using fed-batch and continuous reactors were also by the same group.Using an UV mutant and exponentially feeding a mediumcontaining glucose, urea, phosphates and sulphates, Sharma[133]was able achieve a methionine concentration of 4 g l−1.In a different approach followed by Kumar et al.[134], in-termittent supplementation of cysteine into the media duringmethionine production by a NTG mutantC. lilium M128,resulted in an increase of methionine concentration from2.3 g l−1 to 3.4 g l−1.

Sharma and Gomes[116] reported the production ofl-methionine in a CSTR to determine the dilution rate anddissolved oxygen that would result in the highest amountof methionine and biomass. A UV mutant, resistant to200�g ml−1 norleucine and 4 mg ml−1 triazole, C. liliumNT-33 was used in this study. In shake flasksC. lilium NT-33produced 521�g ml−1 methionine in screening medium. Acentral composite design was used for this purpose and steadystate data obtained from these experiments were analyzed forcombinations of DO and D resulting is high productivity. Thestatistically designed experiments were completed in threecontinuous production runs. The results of one of the contin-us fore as welver state.B e op-t nc le form Piretm

F inuous ate (D)a

.1. Production of methionine in batch, fed-batch andontinuous reactors

Gomes and co-workers[70,116,133]studied of the prouction ofl-methionine in reactors. They developed UVTG mutants ofC. lilium for overproducing methionineubmerged cultivation, and provided a detailed accouhe productivities and genealogy of their mutants. A serieatch reactor studies were carried out using highest me

ig. 4. Dry cell weight (♦), glucose (�) and methionine (�) data for contnd dissolved oxygen (DO)[116].

ous production runs are presented inFig. 4. A minimum ofix reactor volumes was passed for each experiment beteady state was achieved. In some experiments, up to teactor volumes were passed to achieve the steadyased on an analysis of variance, they concluded that th

imum value of dilution of 0.16 h−1 and a dissolved oxygeoncentration of about 40% saturation was most suitabethionine production. They also used a Leudeking–odel to correlate theoretical and experimental results.

reactor experiments carried out at various combinations of dilution r


7.2. Modelling, simulation and control

Since amino acid production is known to be growth associ-ated, the Leudeking–Piret model is normally used to describethese processes. Methionine production is also well describedby this model. The dissolved oxygen concentration plays asignificant role in the production of methionine and has tobe maintained at an optimum level where it is not inhibitory.Consequently, a modification of the Leudeking–Piret modeldeveloped by Pandey[135]and later improved by Garg[136]and Singh et al.[137], which also describes this dependencyon oxygen, is given by the following equation:

dx

dtds

dtdp

dtdcl

dt

=

f1µmsx

Km + s

−f1

(1

Yx/s

+ β

Yp/s

)µmsx

Km + s−

(α

Yp/s

+ ms

)x

αx + βf1µmsx

Km + s

−f1

(1

Yx/o

+ β

Yp/o

)µmsx

Km + s− αx

Yp/o

+

−x 0

(sf − s) 0

−p 0

−cl (c∗l − cl)

[D

kla

](1)

where the biomass concentrationx, residual glucose concen-trations, methionine concentrationp and dissolved oxygenconcentrationcl , constitute the of state variables of the pro-cess. The dilution rateD and the air flow rate included withint -pg rateo rriedo tionst s arem troli comet ly af y, thec vari-a cesse oticc tipleot basedo 15 lr ueso ted inT sesi

8

siss on onm bolicfl con-fi -

ships were based on the work of Park et al.[138]. Other sto-ichiometric relationships and constraints needed to describemethionine production were added to this set. The quantita-tive information, cell mass, residual glucose, methionine, cys-teine, and dissolved oxygen concentrations used in this anal-ysis were obtained from the batch and cysteine supplemen-tation experiments. Other quantitative information requiredwere derived based on a study of carbon flux distribution inC. glutamicumduring lysine overproduction[139,140].

For methionine production, both with and without cysteinesupplementation, the respiratory quotient (RQ) was obtained

as 1.12. Theoretically, the RQ required for maximum methio-nine production was determined to be 0.75. This is less thanhalf the theoretical value of 2.0 required for maximum lysine[ ifieda tiosa then assb noti fluxs nifi-c

ire-m was1 re-m tationr fluxt or-r rvedt ed inF

TV

P s

µ

KYYYYmα

β

K

he oxygen mass transfer coefficientskla, are the external inuts of the process. The functionf1(cl) = cl

Ko+cl(1+(cl/Kio))ives the nonlinear relation between the specific growthn the oxygen dynamics. Simulations are usually caut to predict process behaviour under various condi

hat may be encountered in the industry. Such exerciseeaningful when they are carried out with certain con

mplementations. Even these unstructured models beoo complex to account for oxygen dynamics. Since onew of the state variables can be measured on-line reliablontrol of bioprocesses remains a challenge. Further, thetion of both kinetic and transport parameters during provolution, adds to the complexity of the problem. Asymptonvergence was demonstrated for a multiple input mulutput (MIMO) system resulting from the model[137]. In

he simulations, the process conditions assumed weren actual experimental conditions that would exist in aeactor with a maximum working volume of 12 l. The valf the parameters used in these simulations are presenable 2. A discussion of control strategies for bioproces

s beyond the scope of this review.

. Metabolic flux analysis

Kumar et al.[134] have reported metabolic flux analytudies to elucidate the effects of cysteine supplementatiethionine production in a fed-batch process. The meta

ux analysis was based on the biochemical pathwayrmed to exist inC. glutamicum. The stoichiometric relation

139]. The Glc6P, PEP, Pyr and OaA nodes were idents the principal nodes of the pathway. The flux split rat Glc6P node were changed significantly implying thatode was flexible. However, OaA node was under the malance constraints of the TCA cycle and therefore did

nfluence the pathway fluxes independently. Further, theplit ratios for the PEP and Pyr nodes did not change sigantly indicating that these were rigid nodes.

They have also reported that the energy requent in terms of ATP in the presence of cysteine066 mmol g−1 h−1, which was 20% lower than the requient in the absence of cysteine. Cysteine supplemen

educed the rigidity of the PEP node allowing a highero OaA, thereby increasing the final yield of methionine. Cesponding to the increase in methionine flux, it was obsehat the PPP flux also increased. A flux map is presentig. 5 [134].

able 2alues of model parameters

arameter Value

m (h−1) 0.54

s (g ml−1) 1.0

x/s (g g−1) 0.3

p/s (g g−1) 0.2

x/o (g g−1) 2.33

p/o (g g−1) 0.326

s (h−1) 0.008(g g−1 h−1) 1.0(g g−1) 0.005

io (g ml−1) 0.1


Fig. 5. Metabolic flux analysis showing effect of cysteine on of methionine production by mutant strain. The flux values have been normalized based on100 mmol g−1 h−1of glucose. Flux values for fermentation with cysteine supplementation are given in italics[134]: Glc6P, glucose 6 phosphate; Fru6P,fructose 6 phosphate; GaP, glyceroldehyde 3 phosphate; G3P, 3 phosphoglycerate; PEP, phosphoenol pyruvate; Pyr, pyruvate; AcCoA, acetyl coenzymeA; OaA, oxaloacetate; Isocit, isocitrate; aKG, alpha keto glutarate; SucCoA, succinyl coenzyme A; Suc, succinate; Mal, malate; Asp, aspartate; O-Shs,orthoacetylhomoserine; Meth, methionine; Lys, lysine; Thr, threonine; Ribu5P, ribulose 5 phosphate; Rib5p, ribose 5 phosphate; Xyl5p, xylulose 5phosphate;Sed7P, sedoheptulose 7 phosphate; E4P, erythrose 4 phosphate; ADP, adenosine diphosphate; NADH, reduced nicotinamide adenine dinucleotide; FADH,reduced Flavin adenine dinucleotide; NAD, nicotinamide adenine dinucleotide; FAD, Flavin adenine dinucleotide; ATP, adenosine triphosphate.

9. Recovery of methionine

Methionine can be recovered from fermentation broth byseparating cells and passing the clear broth (pH 5, adjustedusing H2SO4) through activated charcoal (for decolourisa-tion of broth) followed by passing through Amberlite IR-120(H+) under controlled flow rate. The process is repeated un-til no methionine is detected. The column is then washedwith distilled water and methionine is eluted from the resinby treating with 1 N NH4OH. Crystalline methionine canbe obtained from the eluate by concentrating in vacuum,treating with absolute alcohol and keeping overnight at 4◦C[16,22].

10. Conclusion

Methionine is perhaps the only amino acid that has notyet been produced in large quantities or industrially by sub-merged fermentation. It is one of the amino acids in highestdemand and yet there are hardly any reports on the processdevelopment for methionine production. The primary reasonappears to be the difficulty of obtaining high-yield strainsbecause of severe feedback regulation. Among the variousspecies surveyed, it appears thatCorynebacteriumsp. has alesser degree of feedback regulation and that it may be pos-sible to obtain high methionine producingCorynebacteriumstrains. Renewed interest in usingCorynebacteriumsp. for


producing amino acids and to study its metabolic flux maps,may generate new data that would provide an answer to theexisting problems in methionine production. The wider clin-ical applications ofl-methionine observed recently may givethe impetus needed to address this issue.

References

[1] Mueller JH. Proc Soc Exp Biol Med 1922;18:161.[2] Barger G, Coyne FP. Biochem J 1928;22:1417.[3] Vigneaud V, du Chandler JP, Moyer AW, Keppel DM. The effect

of choline on the ability of homocysteine to replace methionine inthe diet. J Biol Chem 1939;131:57–76.

[4] Cantoni GL.S-Adenosylmethionine—a new intermnediate formedenzymatically froml-methionine and adenosinetriphosphate. J BiolChem 1953;204:403–16.

[5] Tabor H, Rosenthal SM, Tabor CW. Biosynthesis of spermi-dine and spermine from putrescine and methionine. J Biol Chem1958;233:907–17.

[6] Adams JM, Capecchi MR.N-Formylmethionyl-sRNA as the initia-tor of protein synthesis. Proc Natl Acad Sci USA 1966;55:147–55.

[7] Clark BFC, Marcker KA. How proteins start. Sci Am1968;218:36–42.

[8] Greenstein JP, Wintz M. Methionine. Chemistry of the amino acid,vol. 3. John Wiley & Sons; 1961. p. 2125–55.

[9] Flavin M. Methionine biosynthesis. In: Greenberg DM, edi-tor. Metabolic pathways. New York: Academic Press; 1975. p.457–503.

. 1.en

-erine-

e

of

is in8.

in-

tial

ion.

iol

onl

ctioni

y

mi-

[21] Morinaga Y, Tani Y, Yamada H.l-Methionine production ethionineresistant mutant of facultative methylotrophPseudomonasFM 518.Agric Biol Chem 1982;46(2):473–80.

[22] Roy SK, Biswas SR, Mishra AK, Nanda G. Production and purifi-cation of methionine from a multianalog reistant mutant B-6 US-215 of Bacillus megateriumB71. J Micro Biotech 1989;4:35–41.

[23] Yamada H, Morinaga Y, Tani Y.l-Methionine overproduction byethionine resistant mutants of obligate methylotroph strain Om 33.Agric Biol Chem 1982;46:47–55.

[24] Aida K, Chibata I, Nakayama I, Takinami K, Yamada H. Biotech-nology of amino acid production. Prog Ind Microbiol 1986:24.

[25] Kircher M, Pfefferle W. The fermentative production ofl-lysineas an animal feed additive. Chemosphere 2001;43(1):27–31.

[26] Lessard PA, Guillouet S, Willis LB, Sinskey AJ. Corynebacteria,brevibacteria. In: Flickinger MC, Drew SW, editors. Encyclopediaof bioprocesstechnology: fermentation, biocatalysis, and biosepara-tion, vol. 2. New York: John Wiley & Sons Inc.; 1999. p. 729–40.

[27] Nakayama K. Amino acids. In: Reed C, editor. Industrial microbi-ology. Westport: AVI Publishers Co.; 1982. p. 748–801.

[28] Okamoto K, Ikeda M. Development of industrially stable processfor l-threonine fermentation by anl-methionine auxotrophic mu-tant of Escherichia coli. J Biosci Bioeng 2000;89(1):87–9.

[29] Umerie SC, Ekwealor IA, Nawabo IO. Lysine production fromvarious carbohydrates and seed meals. Bioresource Technol2000;75:249–52.

[30] Guttormsen AB, Ueland PM, Kruger WD, Kim CE, Ose L, FollingI, et al. Disposition of homocysteine in subjects heterozygous forhomocystinuria due to cystathionine beta-synthase deficiency: re-lationship between genotype and phenotype. Am J Med Genet2001;100:204–13.

[31] Harksen A, Ueland PM, Refsum H, Meyer K. Four common muta-ltiplexflight

H.Clin

Orn-lonicsian

iszctionoti-

J,er-. Biol

AL.

ipidin a

756–

es-Nutr

ids.ed.

anfor

[10] Kinoshita S, Udaka S, Shimono M. Amino acid fermentationProduction ofl-glutamic acid by various microorganisms. J GAppl Microbiol 1957;3:193–205.

[11] Kase H, Nakayama K. Production ofO-acetyl-l-homoserine by methionine analogue resistant mutants and regulation of homosO-transacetylase inCorynebacterium glutamicum. Agric Biol Chem1974;38(10):2021–30.

[12] Kase H, Nakayama K.l-Methionine production by methioninanalog-resistant mutants ofCorynebacterium glutamicum. AgricBiol Chem 1975;39(1):153–60.

[13] Kase H, Nakayama K. Isolation and characterizationS-adenosylmethionine requiring mutants and role ofS-adenosylmethionine in the regulation of methionine biosynthesCorynebacterium glutamicum. Agric Biol Chem 1975;39(1):161–

[14] Kase H, Nakayama K.O-Acetylhomoserine as an intermediatemethionine biosynthesis inArthrobacter paraffineus, Corynebacterium glutamicum and Bacillus species. Agric Biol Chem1975;39(3):687–93.

[15] Nakayama K, Araki K, Kase H. Microbial production of essenamino acid withCorynebacterium glutamicummutants. Adv ExpMed Biol 1978;105:649–61.

[16] Banik AK, Majumdar SK. Studies on methionine fermentatPart I. Selection of mutants ofMicrococcus glutamicusand op-timum conditions for methionine production. Indian J Exp B1974;12:363–5.

[17] Banik AK, Majumdar SK. Effect of minerals on productiof methionine by Micrococcus glutamicus. Indian J Exp Bio1975;13:510–2.

[18] Mondal S, Chatterjee SP. Enhancement of methionine produby methionine analogue resistant mutants ofBrevibacterium heal.Acta Biotechnol 1994;14:199–204.

[19] Mondal S, Das YB, Chatterjee SP.l-methionine production bdouble auxotrophic mutants of an ethionine resistant strain ofBre-vibacterium heali. Acta Biotechnol 1994;14:61–6.

[20] Mondal S, Das YB, Chatterjee SP. Methionine production bycroorganisms. Folia Microbiol 1996;41:465–72.

tions of the cystathionine beta-synthase gene detected by muPCR and matrix-assisted laser desorption–ionization time-of-mass spectrometry. Clin Chem 1999;45(8):1157–61.

[32] McKinley MC, Strain JJ, McPartlin J, Scott JM, McNultyPlasma homocysteine is not subject to seasonal variation.Chem 2001;47:1430–6.

[33] Refsum H, Yajnik CS, Gadkari M, Schneede J, Vollset SE,ing L, et al. Hyperhomocysteinemia and elevated methylmaacid indicate a high prevalence of cobalamin deficiency in AIndians. Am J Clin Nutr 2001;74:233–41.

[34] Kroger H, Dietrich A, Ohde M, Lange R, Ehrlich W, KurpM. Protection from acetaminophen-induced liver damage by aof low doses of the poly(ADP-ribose) polymerase-inhibitor nicnamide and the antioxidantn-acetylcysteine or the amino acidl-methionine. Gen Pharm 1997;28(2):257–63.

[35] Silveri MM, Parow AM, Villafuerte RA, Damico KE, GorenStoll AL, et al. S-Adenosyl-l-methionine: effects on brain bioengetic status and transverse relaxation time in healthy subjectsPsychiatry 2003;54(8):833–9.

[36] Uversky VN, Yamin G, Souillac PO, Goers J, Glaser CB, FinkMethionine oxidation inhibits fibrillation of human�-synuclein invitro. FEBS Lett 2002;517(13):239–44.

[37] George J, Pera N, Phung N, Leclercq I, Hou JY, Farrell G. Lperoxidation, stellate cell activation and hepatic fibrogenesisrat model of chronic steatohepatitis. J Hepatol 2003;39(5):64.

[38] Carroll KK, Kurowska EM. Soy consumption and cholterol reduction: review of animal and human studies. J1995;125(3):594S–7S.

[39] Townsend DM, Tew KD, Tapiero H. Sulfur containing amino acand human disease. Biomed Pharmacother 2004;58(1):47–55

[40] Bedell LS, Hulbert MK. Mosby’s complete drug reference. 7thSt. Louis: Mosby-Year Book Inc.; 1997.

[41] Lewis AJ, Bayley HS. Amino acid bioavailability. In: AmmermCB, Baker DH, Lewis AJ, editors. Bioavailability of nutroientsanimals. San Diego: Academic Press; 1995. p. 35–65.


[42] Baker DH. Amino acid nutrition in pigs and poultry. In: HaresignW, Cole DJA, editors. Recent advances in animal nutrition. London:Butterworths; 1989. p. 249–59.

[43] Lewis LD, Morris ML, Hand MS. Small animal clinical nutrition.3rd ed. Topeka KS: Mark Morris Associates; 1987.

[44] Araki K, Ozeki T. Amino Acids (survey)Kirk-Othmer. Encyclo-pedia Chem Technol 1991;9:504–71.

[45] Fong CV, Goldgraben GR, Konz J, Walker P, Zank NS. Condensa-tion process fordl-methionine production. In: Goldgraben AS, etal., editors. Organic chemicals manufacturing hazards. Ann Arbor,MI: Ann Arbor Science Publishers; 1981. p. 115–94.

[46] Funfstuck R, Straube E, Schildbach O, Tietz U. Prevention of re-infection by l-methionine in patients with recurrent urinary tractinfection. Med Klin 1997;92:574–81.

[47] Gieger F, Halsberghe B, Hasselbach HJ, Hentschel K, HuthmacherK, Korfer M, et al. Process for the preparation ofd,l-methionineor the salt thereof. US Patent No. 5770769; 1998.

[48] Ponf WG, Church DC, Pond KR. Basic animal nutritiion and feed-ing. New York: John Wiley & Sons; 1995.

[49] Cheeke P. Applied animal nutrition, feeds and feeding. 2nd ed.Saddle River, NJ: Prentice Hall; 1999.

[50] Chibata I, Ishikawa T, Yamada S. Studies on amino acids and stud-ies on the enzymatic resolution and specificity of mold acylases.Bull Agric Chem Soc Jpn 1957;21:304–7.

[51] Soda K, Tanaka H, Nakazawa H, Mitsugi K. Japanese Patent 55-1797; 1980.

[52] Tosa T, Mori T, Fuse N, Chibata I. Studies on continuous en-zyme reactions for preparation of a DEAE-sephadex-aminoacylasecolumn and contimuous optical resolution of acyl-dl-amino acids.Biotechnol Bioeng 1967;9:603–8.

[53] Morinaga Y, Tani Y, Yamada H. Homocysteinetrans-methylation

e in

d G,3. p.

cificted

3.n of

ewl

ine-cids.

r

e-4–

tent

s. In:imary

xylicand

zurden

im kreislauf gefuhrten mutterlaugen des kaliumcarbonat-methioininverfahrens. Eur Patent 2421167; 1974.

[65] Mannsfeld SP, Pfeiffer A, Tanner H, Liebertanz E. Continuousprocess for the manufacture of methionine. US Patent 04069251;1978.

[66] Rowbury RJ, Woods DD. Further studies of the repressionof methionine synthesis inEscherichia coli. J Gen Microbiol1961;24:129–44.

[67] Herrmann KM, Somerville RL. Amino acids biosynthesis and ge-netic regulation. Addison-Wesley Publishing Company; 1983. pp.147–244.

[68] Chattopadhyay MK, Sengupta D, Sengupta S. Fermentative pro-duction of methionine by 5-bromouracil resistant mutants ofEs-cherichia coliK-12. Med Sci Res 1995; 23:775.

[69] Tosaka O, Takinami K. In: Aida, et al., editors. Biotechnology ofamino acid production. 1995. p. 152–72.

[70] Kumar D, Garg S, Bisaria VS, Sreekrishan TR, Gomes J. Pro-duction of methionine by a multi-analogues resistant mutant ofCorynebacterium lilium. Process Biochem 2003;38(8):1165–71.

[71] Patte JC, Boy E. Multivalent repression of aspartic semi-aldehyde dehydrogenase inEscherichia coli K-12. J Bacteriol1972;112:84–92.

[72] Patte JC, Bros GL. Regulation of the synthesis of third aspartok-inase and second homoserine dehydrogenase inEscherichia coliK-12. Biochem Biophys Acta 1967;136:245–57.

[73] Rowbury RJ, Woods DD. Repression by methionine of cystathion-ine formation inEscherichia coli. J Gen Microbiol 1964;36:145–8.

[74] Rowbury RJ, Woods DD.O-Succinylhomoserine as an intermedi-ate in the synthesis of cystathionine byEscherichia coli. J GenMicrobiol 1964;36:341–58.

[75] Rowbury RJ. The accumulation ofO-succinylhomoserine byEs-l

and-

Rev

ina

andctions.

andem

tiesM

m

the-

ation.

ho-

Brot

x-2:

in methanol utilizing bacteria and its application tol-methionineproduction. Agric Biol Chem 1984;48:143–8.

[54] Morinaga Y, Tani Y, Yamada H. Biosynthesis of homocysteinfacultative methylotroph.PseudomonasFM 518. Agric Biol Chem1984;47(12):2855–60.

[55] Soda K, Tanaka H, Esaki N. Amino acids. In: Rehm HJ, Reeeditors. Biotechnology, vol. 3. Oxford: Pergamon Press; 198479–530.

[56] Yamashiro A, Kubota K, Yokozeki K. Mechanism of stereospeproduction ofl-amino acids from the corresponding 5-substituhydantoins byBacillus brevis. Agric Biol Chem 1988;52:2857–6

[57] Voelkel D, Wagner F. Reaction mechanism for the conversio5-monosubstituted hydantoins to enantiomerically purel-aminoacids. Ann N Y Acad Sci 1995;750:1–9.

[58] Wagner T, Hantke B, Wagner F. Production ofl-methioninefrom d,l-(2-methylthioethyl) hydantoin by resting cells of a nmutant strain ofArthrobacter speciesDSM 7330. J Biotechno1996;46:63–8.

[59] Homer RJ, Kim MS, LeMaster DM. The use of cystathiongamma-synthase in the production of alpha and chiral amino aAnal Biochem 1993;215:211–5.

[60] Tokuyama S, Hatano K. Overexpression of the gene foN-acylaminoacid racemase fromAmycolatopsissp. TS-1-60 inEs-cherichia coli and continuous production of optically active mthionine by a bioreactor. Appl Microbiol Biotecnol 1996;44:777.

[61] Imi K, Shiozaki T. Process for producing methionine. US Pa05945563; 1999.

[62] Leuchtenberger W. Amino acids-technical production and useRehm HJ, Reed G, Phuler A, Stadler P, editors. Products of prmetabolism. Biotechnology, vol. 6. 1996. p. 492.

[63] Leuchtenberger W, Plocker U. Amino acids and hydroxycarboacid. In: Gerhartz W, editor. Enzymes in industry, productionapplication. Weinheim: VCH; 1990.

[64] Lussling T, Muller K, Schreyer G, Theissen F. Verfahrengewinnung von methionin und kaliumhydrogencarbonat aus

cherichia coli and Salmonella typhimurium. J Gen Microbio1964;37:171–80.

[76] Stadtman ER, Le BG, De Robichon SH. Feedback inhibitionrepression of aspartokinase activity inEscherichia coliandsaccharomyces cerevisiae. J Biol Chem 1961;236:2033–8.

[77] Umbarger HE. Regulation of amino acid metabilism. AnnBiochem 1969;38:323–49.

[78] Yugari Y, Gilvarg C. Co-ordinated end product inhibitionlysine synthesis inEscherichia coli. Biochem Biophys Act1962;62:612–4.

[79] Wijesundra S, Woods DD. The catabolism of cystathionine byEs-cherichia coli. J Gen Microbiol 1962;29:353.

[80] Flavin M, Devalier-Klutecko C, Slaughter C. Succinic esteramide of homoserine: some spontaneous and enzymatic reaScience 1964;143:50–2.

[81] Kerr DS, Flavin M. The regulation of methionine synthesisthe nature of cystathionine�-synthase in neurospora. J Biol Ch1970;245:1842–55.

[82] Morinaga Y, Tani Y, Yamada H. Regulatory properof l-methionine biosynthesis in obligate methylotroph O33: role of homoserine-O-transsuccinylase. Agric Biol Che1982;46(1):57–63.

[83] Rukert C, Puhler A, Klinowski J. Genome-wide analysis ofl-methionine biosynthetic pathway inCorynebacterium glutamicum by targeted gene deletion and homologous complementJ Biotechnol 2003;104:213–28.

[84] Wyman A, Paulus H. Purification and properties ofmoserine transacetylase fromBacillus polymyla. J Biol Chem1975;250:3897–903.

[85] Bachmann BJ. Linkage map ofEscherichia coliK-12. MicrobiolRev 1983;47:180–230.

[86] Shoeman R, Redfield B, Coleman T, Greene RC, Smith AA,N, et al. Regulation of methionine synthesis inEscherichia coli:effect of metJ gene product andS-adenosylmethionine on the epression of themetF gene. Proc Natl Acad Sci USA 1985;83601–5.


[87] Shoeman R, Redfield B, Coleman T, Greene RC, Smith AA, Saint-Girons I, et al. Regulation of methionine synthesis inEscherichiacoli: effect of metJ gene product andS-adenosylmethionine onthe in vitro expression ofmetB, metC and metJ genes. BiochemBiophys Res Commun 1985;133:731–9.

[88] Su CH, Greene RC, Holloway CT. Regulation ofS-adenosylmethionine synthetase inEscherichia coliK-12. BacteriolProc 1970;70:136–9.

[89] Greene RC, Hunter JSV, Coch EH. Properties ofmetK mutants ofEscherichia coliK-12. J Bacteriol 1973;115:57–67.

[90] Urbanowski ML, Stauffer LT, Plamann LS, Stauffer GV. A newmethionine locus metR that encodes a trans acting protein requiredfor activation of metE and metH inEscherichia coliandSalmonellatyphimurium. J Bacteriol 1987;169:1391–7.

[91] Cai XY, Maxon ME, Redfield B, Glass R, Brot N, WeissbachH. Methionine synthesis inEscherichia coli: effect of the metRprotein on metE and metH expression. Proc Natl Acad Sci USA1989;86:4407–11.

[92] Maxon ME, Redfield B, Cai XY, Shoeman R, Fujita K, Fisher W,et al. Regulation of methionine synthesis inEscherichia coli: effectof the metR protein on metE and metH expression. Proc Natl AcadSci USA 1989;86:85–9.

[93] Kung HF, Spears C, Greene RC, Weissbach H. Regulation of theterminal reactions in the methionine biosynthesis by Vitamin B12

and methionine. Arch Biochem Biophy 1972;150:23–31.[94] Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkowasky

A, et al. The completeCorynebacterium glutamicumATCC 13032genome sequence and its impact on the production ofl-aspartatederived aminoacids and vitamins. J Biotechnol 2003;104:5–25.

[95] Fan C, Chen L, Zheng S. Characteristics ofl-lysine high yielding

cing

t

f-

Ef-i-tt

ro-36;

ro-996;

on-me-

i J,

–

s ofol

d.

in-e

[107] Ghosh BB, Banerjee AK. Production of methionine and glutamicacid from n-alkanes bySerratia marcescensvar. kiliensis. FoliaMicrobiol 1986;31:106–12.

[108] Chattopadhyay MK, Ghosh AK, Sengupta S, Sengupta D, SenguptaS. Threonine analogue resistant mutants ofEscherichia coliK-12.Biotechnol Lett 1995;17:567–70.

[109] Nakamori S, Kobayashi T, Nishimura H, Takagi H. Mechanismof l-methionine overproduction byEscherichia coli: the replace-ment of Ser-54 by Asn in the Metj protein causes the derepressionof l-methionine biosynthetic enzymes. Appl Microbiol Biotechnol1999;52:179–85.

[110] Odessa State University. New strain ofPseudomonas putida. Rus-sian Patent 1730152; 1992.

[111] Genencor. Production of methionine and homoserine by fermenta-tion. US Patent 9317112; 1993.

[112] Halasz A, Szakacs-Dobozi M, Bruschi M. Poly-met DNA yeasthybrids: construction and characterisation. In: Proceedings of the17th International Conference on Yeast Genetics and MolecularBiology. 1995.

[113] Fotheringham I. Method for hypersecretion of amino acid. USPatent 5354672; 1994.

[114] Martinez-Force E, Benitez E. Amino acid overproduction andcatabolic regulation inSaccharomyces cerevisiae. Biotechnol Prog1994;10:372–6.

[115] Ward OP. Fermentations raw materials. In: Fermentation biotech-nology: principles, processes and products. Saddle River, NJ: Pren-tice Hall; 1989. p. 59–71.

[116] Sharma S, Gomes J. Effect of dissolve oxygen on continuous pro-duction of methionine. Eng Life Sci 2001;1:69–73.

[117] Pham CB, Galvez CF, Padolina WG. Methionine production bybatch fermentation from various carbohydrates. ASEAN Food J

f

ch-

-

onol

n of

hro-4–6.n-

J

ane-manatogr

ofckel

or-ino

455–

e-o-

nceeter-

Anal

strain FMC8611. Weishengwuxue Zashi 1988;8:11–7.[96] Hirao T, Nakano T, Azuma T, Nakanishi T. Process for produ

l-lysine. Eur Patent 327945; 1989.[97] Nakano T, Azuma T, Kuratsu Y. Process for producingl-lysine

by iodothyronine resistant strains ofC. glutamicum. US Paten5302521; 1994.

[98] Tosaka O, Yoshihara Y, Ikeda S, Takinami K. Production ol-lysine fluoropyruvate sensitive mutants ofBrevibacterium lactofermentum. Agric Biol Chem 1985;49:1305–12.

[99] Hadj-Sassi A, Colello N, Deschamps AM, Cebeault JM.fect of medium composition onl-lysine production by a varant of Corynebacterium glutamicumATCC 21513. Biotechnol Le1990;12:295–8.

[100] Yamada K, Tsutsui H, Yotsumoto K, Takeuchi M, Shirai M. Pcess for producingl-threonine by fermentation. Eur Patent 21351987.

[101] Kino K, Okamoto K, Takeda Y, Kuratsu Y. Process for the pduction of amino acids by fermentation. Eur Patent Appl 5571993.

[102] Cherest H, Surdin-Kerjan Y, Antoniewski J, de RobichSzulmajster H.S-Adenosyl methionine mediated repression ofthionine biosynthetic enzymes inSaccharomyces cerevisiae. J Bac-teriol 1973;115:1084–93.

[103] Morzycka E, Sawnor-Korszynska D, Paszewski A, GrabskRaczynska-Bojanowska K. Methionine over production bySac-charomyces lipolytica. Appl Environ Microbiol 1976;32:12530.

[104] Komatsu K, Yamada T, Kodaira R. Isolation and characteristicpool methionine rich mutants of aCandidaSp. J Ferment Techn1974;52:93–9.

[105] Tani Y, Lim WJ, Yang HC. Isolation ofl-methionine-enrichemutants of a methylotrophic yeast,Candida boidinii No. 2201J Ferment Technol 1988;66:153–8.

[106] Brigidi P, Matteuzzi D, Fava F. Use of protoplast fusion totroduce methionine overproduction intoSaccharomyces cerevisia.Appl Microbiol Biotechnol 1988;28:268–71.

1992;7:34–7.[118] Avram D, Stan R, Vassu T, Vamanu A, Dan F. Isolation ol-

methionine enriched mutants from methylotrophic yeastCandidaboidinii ICCF26 in a sulfur deficient medium. Recent Adv Biotenol 1966:495–6.

[119] Hilliger M, Hanel F. Process analysis ofl-lysine fermentation under different oxygen supply. Biotechnol Lett 1991;3:219–24.

[120] Akashi K, Shibai H, Hirose Y. Effect of oxygen supplyl-lysine, l-threonine andl-isoleucine fermentations. Agric BiChem 1979;43:2087–92.

[121] Gehrke CW, Neuner TE. Automated chemical determinatiomethionine. J Assoc Official Anal Chem 1974;57(3):682–8.

[122] Shtutman TM. Quantitative determination of methionine on cmatograms using iodoplatinate. Laboratornoe Delo 1966;5:28

[123] Ellinger GM, Duncan A. The determination of methioine in proteins by gas liquid chromatography. Biochem1976;155(3):615–21.

[124] Shinohara Y, Hasegawa H, Tagoku K, Hashimoto T. Simultous determination of methionine and total homocysteine in huplasma by gas chromatography–mass spectrometry. J ChromB: Biomed Sci Appl 2001;758(2):283–8.

[125] Ivanov CP, Alexiev BV, Krsteva M, Yordanov B. Determinationmethionine in protein hydrolysates by reaction with raney niand infrared spectroscopy. Anal Chem Acta 1964;30:549–55.

[126] Larry N, Mackey N, Terri A, Beck. Quantitative high perfmance liquid chromatographic determination of sulphur amacids in protein hydrolysates. J Chromatogr 1982;240(2):61.

[127] Scislowski PWD, Harris I, Pickard K, Brown DS, Buchan V. Dtermintion of l-methionine-dl-sulfoxide in tissue extract. J Chrmatogr 1993;619(2):299–305.

[128] Wagner J, Claverie N, Danzin C. A rapid high performaliquid chromatographic procedure for the simultaneous dmination of methionine, ethionine,S-adenosylmethionine,S-adenosylethionine and natural polyamines in rat tissue.Biochem 1984;140(1):108–16.


[129] Fink K, Cline RE, Fink RM. Anal Chem 1963;35:389.[130] Jones BN, Gilligan JP.O-Pthalaldehyde precolumn derivatisation

and reversed phase high performance liquid chromatographyofpolypeptide hydrolysates and physiological fluids. J Chromatogr1983;266:471–82.

[131] Herbert G, Theodor G, Peter F, Peter P, Peter F. Measure-ment of free amino acids in human biological fluids by highperformance liquid chromatography. J Chromatogr 1984;297:49–61.

[132] Qureshi G, Fohlin L, Bergstrom J. Application of high per-formances liquid chromatography to the determination of freeamino acids in physiological fluids. J Chromatogr 1984;297:91–100.

[133] Sharma S. Strain improvement for the production of methionine.Ph.D. thesis, IIT Delhi, India; 2000.

[134] Kumar D, Subramanian K, Bisaria VS, Sreekrishnan TR, GomesJ. Effect of cysteine on methionine production by a regulatorymutant ofCorynebacterium lilium. Bioresource Technol, 2005; 96:287–294.

[135] Pandey S. Comparison of control strategies forl-methionine pro-duction by Corynebacterium lilium, M. Tech. thesis, IIT Delhi,India; 2001.

[136] Garg S. Development of a nonlinear controller for the productionof l-methionine by a mutant strain ofCorynebacterium lilium, M.Tech. thesis, IIT Delhi, India; 2002.

[137] Singh R, Dhingra SC, Gomes J. Nonlinear geometric controller forbioprocesses: application to methionine production. In: Proceedingsof the International Conference on Chemical and Bioprocess En-gineering. 2003. p. 917–22.

[138] Park SM, Sinskey AJ, Stephanopoulos G. Metabolic and physiolog-ical studies ofCorynebacterium glutamicummutants. BiotechnolBioeng 1997;55(6):864–79.

[139] Vallino JJ, Stephanopoulos G. Metabolic flux distribution inCorynebacterium glutamicumduring growth and lysine overpro-duction. Biotechnol Bioeng 1993;41:633–46.

[140] Vallino JJ, Stephanopoulos G. Carbon flux distribution at the pyru-vate branch point inCorynebacterium glutamicumduring lysineoverproduction. Biotechnol Prog 1994;10(3):320–6.

[141] Kitamoto HK, Nakahara L. Isolation of anl-methionine enrichedmutant of Kluyvermyces lactisgrown whey permeate. ProcessBiochem 1994;29:127–31.

[142] Roy SK, Mishra AK, Nanda G. Extracellular production ofl-methionine byBacillus megateriumB71 isolated from soil. CurrSci 1984;53(24):1296.

production of l-methionine by submerged fermentation: a review

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