The production of l -lysine by fermentation

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Proc_e__ss " Spoflight

fermentum 9. The intracellular pool of Lthreonine in these organisms is considerably reduced and they are capable of accumulating 13-34 g of L-lysine per litre.

Regulatory mutants Another effective technique for the overproduction of amino acids is the use of regulatory mutants. The key to this method is to obtain mutants which are insensitive to feedback inhibition or Osamu Tosaka, Hitoshi Enei and Yoshio Hirose repression. Such organisms have been isolated as mutants which are resistant O v e r 40 000 tons o f L-lysine are used each year, chiefly as a nutritional to amino acid analogs, or as revertants s u p p l e m e n t in a n i m a l feeds. T w o m a j o r biotechnological processes are derived from the auxotroph deficient in regulatory enzymes. A typical example used to m a n u f a c t u r e lysine: the e n z y m a t i c conversion Of D L - ~ a m i n o - f c a p r o l a c t a m , and the direct f e r m e n t a t i o n of m o l a s s e s or starch by is an L-lysine producer derived from m i c r o b e s , especially species o f Brevibacteria and Corynebacterium glu- B. flavum '°,11. The regulation of L-lysine biosynthetamicum. This r e v i e w focuses on the f e r m e n t a t i v e p r o c e s s e s and dessis in B. flavum is less complicated than cribes the d e v e l o p m e n t o f various m u t a n t s which o v e r - p r o d u c e L-lysine. that in E. coli (Fig. 2): only one species ofaspartokinase is subject to a concertAnimal feeds such as grain and defatted this aspect oflysine production that we ed feedback inhibition by L-lysine and oil seeds contain only small quantities focus. L-threonine in B. flavum. Growth of of L-lysine. Since poultry, pigs, cattle this organism is inhibited by an analog and other livestock are unable to Direct f e r m e n t a t i v e production o f of lysine, S-(2-aminoethyl) L-cysteine synthesize this amino acid it must be L-lysine (AEC). This inhibition is markedly added to these feedstuffs to provide an The biosynthesis of amino acids in enhanced by L-threonine, but reversed adequate diet. The increasing microorganisms is normally regulated by L-lysine. This implies that AEC economic importance of L-lysine is to meet the organism's needs, but behaves as a false feedback inhibitor of illustrated by Fig. 1 which shows that artificial distortions of metabolism can aspartokinase. Regulatory mutants in by 1981 over 40 000 tons oflysine were result in the overproduction of parti- which aspartokinase is insensitive to manufactured each year, with a value of cular amino acids. A great variety of the concerted feedback inhibition were $160 million. The majority of this is microorganisms have been reported to expected to be found among mutants produced by Japanese companies or by overproduce L-lysine, including auxo- resistant to AEC. overseas companies operating under trophic mutants and regulatory One such mutant, FA1-30, produces licences issued by Japanese firms. In mutants (Table 1). In addition to the 31-33 g per litre of L-lysine; its aspartoaddition to its use as a feed supplement, conventional mutation techniques used kinase is approximately 150-fold less L-lysine finds pharmaceutical to obtain these strains, protoplast sensitive to feedback inhibition by Lapplications in the formulation of diets fusion and recombinant DNA tech- lysine plus L-threonine than the parent with balanced amino acid compositions nologies have recently been introduced strain. and in amino acid infusions. into the breeding oflysine producers. Two biotechnological processes are now employed commercially to manu- .4 uxotrophic mutants facture L-lysine: direct fermentation by L-Lysine was the first amino acid to 40 bacteria and enzymatic conversion of be produced on an industrial scale with DL-a-amino-~-caprolactam into L- the aid of auxotrophs when homolysine. Microbial processes using serine-requiring auxotrophic mutants -~ molasses or starch hydrolysates as raw of Corynebacterium glutamicum were ~ 30 material have become firmly estab- derived as L-lysine producers 13. It was o o lished ~s the major methods for found that, in the genera Coryneproducing L-lysine, having replaced bacterium and Brevibacterium, asparto- vX2 0 methods based on the hydrolysis of kinase catalyses the first step in the synproteins. The development of better thesis of L-lysine and L-threonine from strains of microbe to produce lysine has aspartate, and that this reaction is had a major impact on the economics of subject to a concerted feedback inhibi- ~ 10 the fermentation process and it is on tion by L-lysine plus L-threonine. Hence, a homoserine auxotroph and a Osamu Tosaka, Hitoshi Enei and Yoshio threonine-methionine double auxoHirose are all at the Biochemistry Depart- troph were isolated from glutamate 196~ 1670 1975 1~0 ment, Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-Cho, producing strains of C. glutamicum, Brevibacterium flavum 4 and B. lacto- Fig. 1. World supply of L-lysine. Kawasaki-Ku Kawasaki, Japan.

The production of L-lysineby fermentation

@ 1983,ElsevaerScxencePubhshersBN,Pansterdam 0166-9430183l$0100

Trends in Biotechnology, Vol. 1, No. 3, 1983

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Better producers of L-lysine were obtained by repeated mutation. Table 2 shows the genealogy of L-lysineproducing mutants of B. lactofermenturn and their productivity. The lysine productivity was improved stepwise by successive mutations which endowed the bacteria with resistance to AEC, a requirement for alanine, resistance to a-chlorocaprolactam (CCL) and 7methyl-L-lysine (ML) and sensitivity to /3-fluoropyruvate (FP). One AEC-resistant mutant, AJ3445, produced 16 g per litre of L-lysine12,13. In this strain, the intracellular pool of DL-alanine was greater than that of the other amino acids. It was known that alanine and lysine were formed from pyruvate and aspartate, and, therefore, alanine auxotrophs were expected to produce more L-lysine than the parent strain. In fact, an alanine auxotroph derived from a AEC-resistant mutant produced 33 g of L-lysine per litre15. The growth and aspartokinase activity of AEC-resistant, alanine-requiring, mutant AJ3424 were still sensitive to other lysine analogs, CCL and ML. A mutant resistant to these lysine analogs, derived from AJ3424, produced 60 g of L-lysine per litre, with a conversion yield of 43% from glucose '6. Excess biotin (200-500/ag per litre) stimulated the formation of L-lysine and the specific incorporation of CO2 into the),CH2 group ofL-lysine17.Both pyruvate carboxylase and phosphoenolpyruvate carboxylase were found in the extract of B. lactofermentum AJ399 l~S. These findings indicate that oxaloacetate production is stimulated by excess biotin, resulting in the promotion of L-lysine production. When the optimal level of pyruvate dehydrogenase activity was maintained in L-lysine biosynthesis, pyruvate was preferentially converted to oxaloacetate by the biotin-dependent enzyme pyruvate carboxylase 19. With this in mind, a fluoro-pyruvate sensitive mutant which had an optimal level of pyruvate dehydrogenase activity was bred from AJ39912°. The best producer, AJl1214, accumulated 70 g of L-lysine per litre, with a conversion yield of 50% from glucose in the presence of excess biotin. Furthermore, it was found that citrate synthase plays an important role in the conversion of oxaloacetate to L-lysinCL

Table 1. Production of L-lysineby mutants Microorganism

Genetic markers

Accumulation oflysine-HCl (g/litre)

Re£

Auxotrophic mutants

Corynebacterium glutamicum Brevibacterium d#laz~ul,?l Breoibaaerium

Homoser-

13

2

Thr-Met-

34

4

ThrSMet s

25

5

AEC r

32

11

AECrAHV r

29

12

AECr

3.2

6

AEC r

1.8

7

AEC r

0.03

8

AECrAIa- CCLrML r

60

16

AECrAIa-CCLrMLrFps

70

20

AECrHomoser-LeuPant-

42

14

Regulatory

mutants

Auxotrophic

regulatory mutants

flavum

Brevibacterium flavum Brevibacterium lactofermentum Candida pelliculosa Pseudomonas acidovorans Escherichia coli Brevibactenum

lactofermentum Brevibacterium lactofermemum Corynebacterium glutamicum

AEC, S-q3-Aminoethyl)L-cysteine;AHV,a-Amino-/3-hydroxyvalericacid; CCL, a-Chlorocaprolactam; ML, y-Methyl-L-lysine; FP,/3-Fluoropyruvate; Pant, Pantotheic acid. Superscripts: r, resistant to compound indicated; - , requires compound indicated; s, sensitive to compound indicated.

Asp

,Thr

ASA~Hse

U--

lie M!t

1_

Escherichia coil

Asp

.

'

ASA

" Hse

"

" Thr

,!e

DDP

;

DAP

~, i

Protoplast fusion techniques

Mutant

Enzyme reactton Feedback inhibition

Brevibacterium flavum <~---] Repression

Fig. 2. Regulationoflysine biosynthesisin E. coil and B. flavum. ASA, aspartate-fl-semialAlthough these successive mutations dehyde; DDP, dihydrodipicolinate.DAP, a, e-Diaminopimelate;Hse, homoserine.

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/uns

Table 2. Genealogy of L-lysine-producing mutants and their productivity Yield of L-lysine• HCI(%) AJ1511(wild) 0 AJ3445(AEC~ 16 AJ3424(AECr,Ala-) 33 AJ3796(AEC~,Ala-, CCL) 39 AJ3991(AECr, Ala-, CCLr, MLr) 43 AJI 1214(AECr,Ala-, CCL~,MLr,FP') 50

Wild strain

Brevibacteriurnlactofermentum

AEC, S-(/3-aminoethyl)L-cysteine; CCL, achlorocaprolactam;ML, y-methyl-L-lysine;FP, fl-fluoropymvate. Superscripts: r, resistant; -, requiring; s, sensitive.

( ,,sproc,on.) lucose consumption rate

)

,~. glucose consumption rate

)

Fusion

did increase lysine production, the overproducing strains often showed lower rates of glucose consumption and ~ Lys production, • . glucose consumpgrowth. Tosaka et al.52 demonstrated tion rate the advantages of protoplast fusion in B. lactofermentum to improve the glucose consumption rate of these Fig. 3. Strategy for improving glucose consumption rate of L-lysine producers. lysine producers (Fig. 3). One of the two strains fused was a glutamic acid Escherichia coli strain, TOCR21, that (aspartokinase III) and the lysA gene producer, resistant to decoyinine overproduces L-lysine because of a (DAP decarboxylase) and trying to (DC)* and ketomalonate (KM), which mutation in its aspartokinase2L reduce the activity of inducible lysine was able to consume glucose rapidly. TOCR21 and the plasmid-habouring decarboxylase. However, the producThe other was the lysine producer strain accumulated 4 and 6.5 g of L- tion of L-lysine by these strains ofE. coli AJl1214 with a lower glucose con- lysine per litre, respectively. Only was still much lower than of the analogsumption rate. Resistance to AEC, DC plasmids which carried the dap A gene resistant mutants derived from Breviand K M was used as the selection (encoding dihydrodipicolinate synthe- bacterium and Corynebacterium. marker. One strain among fusants con- tase) caused an increase in L-lysine sumed 130 g of initial glucose per litre production. This clearly indicates that Production conditions in 30 h as compared with 100 h for the the limiting step in the synthesis of To reproduce the results of laboraparent lysine producer and it produced lysine in E. coliis the condensation step tory-scale fermentation in large 3 times as much lysine. catalysed by this enzyme. Lebeault et commercial operations (Fig. 4), a al. 28attempted to further improve this number of biochemical engineering Recombinant D N A technology strain by amplifying the lysC gene problems must be solved: pure cultures A major aim of recombinant DNA technology in the amino acid fermentation industry is to increase the number Microorganisms Raw materials of genes which encode the key enzyme(s) for the biosynthesis of an Brevibacterium Tapioca flour --->glucose amino acid. Such an increase in the lactofermentum Beet or cane molasses number of genes (carried on plasmids) would lead to the overproduction of an amino acid if the synthesis of the , (Continuous sterilization) relevant enzyme(s) increases with the gene copy number and if the reaction it 5t t catalyses is one of the limiting steps in (Fermentation) the synthesis of this amino acid. Nutrients The principle of gene amplification Fermentation broth Soy bean hydrolyzate has already been developed by Sano23 L-Lys" HC170 g/htre Vitamins and Miwa 24 for L-lysine fermentation Production yield 50% Salts and by Devabov25 for L-threonine B fermentation. Richaud et al. 26 has cloned several genes of the lysine bio(Isolation) synthetic pathway on the high copyI Air filter ~--T number plasmid pBR322. These hybrids were used to transform an Lys • HCI Ammonia I

)

1

I

'

/

t

*Decoyinine~ angustmycinA = 6-amino-9-{LFig. 4. Flow diagram of industrial L-lysinefermentation• 1,2-fucopyranoseenyl)-purine.

Trends in Biotechnology, VoL 1, No. 3, 1983

73

D-Aminolactam 0

Success in these endeavours will ensure that the L-lysine industry continues to grow in economic importance.

C -OH

References

JIRacemase(Achromobacterobae)

/

N

,C

\

/

\

1 Kinoshita, S., Nakayama, K. and Kitada, S. (1958),7. Gen. Appl. Microbiol. 4, 128-129 CH-NH 2 2 Nakayama, K., Kitada, S. and Kinoshita, S. (1961) J. Gen. Appl. / Hydrolas~ Microbiol. 7, 145-154 3 Nakayama, K., Tanaka, H., Hagino, H. and Kinoshita, S. (1966)Agric. Biol. Chem. 30, 611-616 4 Shiio, I. and Sano, K. (1969) J. Gen, Appl. Mierobiol. 15, 267-287 5 Shiio, I. and Sano, K. (1969).7. Gen. Fig. 5. Enzymatic synthesis of L-lysine from D-arninolactam. Appl. Microbiol. 15, 267-287 6 Takenouchi, E., Yamamoto, T., Nikolova, D. K., Tanaka, H. and Soda, must be maintained; the fermentation Enzymatic synthesis of L-lysine K. (1979) Agrie. Biol. Chem. 43, broth must be aerated and agitated; and Fukumura 32 has established an 727-734 pH, temperature and foaming must be enzymatic process for production of L7 Hermann, M., Thevenet, N. J., controlled. lysine using DL-a-amino-e-caprolactam Coudert-Maratier, M. M. and Molasses or starch hydrolysate are (AAC) as a starting material (Fig. 5). Vandecasteele, J-P. (1972) Eur. J. now generally used as carbon sources. This process is composed of two Biochem. 30, 100 The optimum fermentation tempera- enzymatic reactions: the selective hy- 8 Halsall, D. M. (1975) Biochem. Genet. ture is 32°C and the pH is maintained drolysis of L-AAC to L-lysine, and the 13, 109-124 near neutrality during the fermentation racemization of AAC. The L-AAC hy- 9 Tosaka, O., Hirakawa, H. and by feeding ammonia. The supply of drolysing enzyme was obtained from Takinami, K. (1979)Agric. Biol. Chem. oxygen is particularly important in L- the cells of Cryptococcus laurendi~ and 43, 491-495 lysine fermentations. its synthesis was induced by DL-AAC. 10 Sano, K. and Shiio, I. (1970)J. Gen. Appl. Microbiol. 16, 373-391 The biosynthesis of L-lysine is an AAC racemase was found in the cells of aerobic process and Akashi et al. 29 Achromobacter obae. Ahmed et al. 33 11 Sano, K. and Shiio, I. (1971),7. Gen. Appl. Mierobiol. 17, 97-113 elucidated the relationship between purified and characterized this enzyme. 12 Tosaka, O.,Taldnami, K.andHirose,Y. oxygen supply and product formation 100 g per litre of DL-AAC can be con(1978)Agrie. Biol. Chem. 42, 745-752 in lysine fermentations. The maximum verted into L-lysine in 25 h with a yield 13 Tosaka, O., Takinami, K. and Hirose, accumulation of L-lysine was observed of 99.8% (moles of product per mote of Y. (1978)Agric. Biol. Chem. 42, when the cells' requirements for substrate). The Japanese firm Toray 1181-1186 oxygen respiration are satisfied; a produces L-lysine by this route, and the 14 Nakayama, K. and Araki, K. (1981) limited oxygen supply results in the annual production of lysine from DL-' Japan patent 56-8692 accumulation of lactic acid. AAC was reported to exceed 4000 tons 15 Tosaka, O., Hirakawa, H., Yoshihara, Gr~ife et al. 3° also reported that in 1981. Y., Takinami, K. and Hirose, Y. (1978) Agric. Biol. Chem. 42, 1773-1778 oxygen depletion irreversibly inhibited 16 Kubota, K., Tosaka, O., Yoshihara, Y. the production of L-lysine by a homo- Future prospects and Hirose, Y. (1976)Japan patent The demand for L-lysine in foods, serine auxotroph ofC. glutamium. This 51-19186 animal feeds and pharmaceuticals is inhibition was accompanied by an increase in the intracellular pools of L- still increasing. To meet this demand 17 Tosaka, O., Hirakawa, H. and Takinami, K. (1979)AgHc. Biol. Chem. lysine and phospholipid. This suggests the amino acid industry is attempting to 43, 491-495 that the reduction of Lqysine improve the l~roduction technology by 18 Tosaka, O., Morioka, H. and accumulation under these conditions reducing costs and utilizing unusual reTakinami, K. (1979)Agric. Biol. Chem. was due to an alteration of the cell mem- sources. Promising approaches for im43, 1513-1519 brane which decreases the permeability proving the productivity of lysine fer- 19 Tosaka, O., Morioka, H. and mentation in future include: of L-lysine. Takinami, K. (1979)Proc. Annu. Meet. Ruklisha et al. 31 formulated the (1) Breeding better lysine producers . Agric. Chem. Soc. Jpn., p. 176 material and energy balances for the with the aid of genetic engineering 20 Tosaka, O., Morioka, H. and Takinami, K. (1981) U.S. patent main pathway of lysine biosynthesis and protoplast fusion. 4275157 from glucose in B. flavum. The (2) Introducing less expensive raw 21 Tosaka, O., Ikeda, S., Yoshii, H. and maximum accumulation of L-lysine materials. Shiio, I. (1981) Japan patent 56-88799 was obtained at 0.40-0.05 atm of PL 22 Tosaka, O., Karasawa, M., Ikeda, S. (liquid phase oxygen tension). This (3) Optimizing the physical and metaand Yoshii, H. (1982) Proc. 4th Int. bolic culture conditions. condition induced the maximum level Symp. Genet. Ind. Microorg. p. 61 of PEP carboxylase and isocitrate (4) Combining microbial processes 23 Sano, K. and Tsuchida, T. (1981) dehydrogenase activities. with chemical synthesis. Japan patent 56-18596

H2C~

\

/

/CH2 H2C~ c / C H 2 2 (Cryptococcuslaurentl0 H2 L-Aminolactam L-Lysine

Trendsin Biotechnology, Vol.1, No. 3, 1983

74

Sano, K., Takinami, K. and Patte, J-C. Biotechnology Letters. 3, 461-464 24 Miwa, K., Terabe, M., Ishida, M., (1982) Eur. J. Appl. Microb. Biotechnol. 31 Ruklisha, M., Marauska, D., Shivinka, Matsui, H. and Momose, H. (1981) 15, 227-231 Japan patent 56-160997 J., Toma, M. and Galynina, N. (1981) 25 Debabov, V. G. (1982) Proc. 4th Int. 28 Lebeual, J. M. and Patte, J-C. (1982) Biotechnol. Lett. 3, 465-470 Proc. 4th Int. Symp. Genet. Ind. Symp. Gent. Microorg. p. 9 32 Fukumura, T. (1977)Agric. Biol. Microorg. p. 113 26 Richaud, F., Richaud, C., Haziza, C. Chem. 41, 1327-1330 and Patte, J-C. (1981) C. R. Acad. Sc. 29 Akashi, K., Shibai, H. and Hirose, Y. (1979) Agric. B i o L Chem. 43, 33 Ahmed, S. A., Esaki, N., Soda, K. and Paris sdrieIII 293, 507-512 Yotsumoto, K. (1981) Seikagaku (J. 2087-2092 27 Reverend, B. D., Boitel, M., Japan. Biochem. Soc.) 53, 841 Deschamps, A. M., Lebeanlt, J-M., 30 Hilliger, M. and H~nel, F. (1981)

Advantages of using thermophiles in biotechnological processes: expectations and reality Bemhard Sonnleitner and Armin Fiechter When thermophilic organisms were first considered for use in biotechnology, certain advantages were expected. The extraordinarily high reaction rates and insensitivity of processes to contaminations have not been experienced in practice, but over the last decade increasing interest has been shown in the possibility of deriving a wide variety of bioproducts from thermophiles. In addition to their high thermo. stability they also promise to have greater tolerance to organic solvents and a longer useful life. The possibility of recovering volatile products directly from a culture provides the opportunity to develop simplified, elegant bioprocesses. However, a series of engineering problems remain to be solved. Thermophilic organisms The term 'thermophilic' is rather illdefined and since it is central to this article, it must be specified further. During the last decade the expression 'thermophilic' has been increasingly restricted to organisms which can grow and/or form products at temperatures ~65°C. In 1953, Allen described at least six different species of the genus Bacillus as being 'thermophilic'. In Bergey's Manual of Determinative Bacteriology, 1975, however, Gibson and Gordon accepted B. stearothermophilus as the only true thermophilic Bernhard Sonnleitner and A r m i n Fiechter a r e a t the Department of Biotechnology, Swiss Federal Institute of Technology, ETH-Ziirich Hoenggerberg, C H 8093 Z i i r i c h , Switzerland.

species in this genus. B. coagulans, which has a maximal growth temperature of below or around 65°C, was classified as 'thermotolerant'. Growth or product formation at this temperature is currently used to distinguish thermophiles and non-thermophiles. If growth occurs at temperatures ) 7 5 ° C the respective organism is termed 'extremely thermophilic' or 'caldoactive'. However, this terminology is likely to be revised in the near future, since organisms capable of growth at temperatures exceeding 100°C have been found in the last few years (see Table 1). Many organisms that can be cultivated at temperatures only little over 37°C are also often named 'thermophilic', for instance several strains ofB. subtilis or B. licheniformis, because they are often used for the

© 1983,ElsewerSciencePubhshersB.V.,Amsterdam0166-9430/83/$01.00

production of thermostable technical enzymes. Many thermophilic organisms are known today, among them bacteria, algae and fungi. The group of true thermophiles, i.e. organisms that require high temperatures and are not active at ambient temperatures, comprises only a few species of eubacteria and most of the species now known as archaebacteria. Among them are strains of Bacillus, Thermus, Clostridium, Thermoanaerobacter, Thermoanaerobium, Thermobacteroides and of Methanobacteriales, Sulfolobales, Thermoplasmales, and Thermoproteales. They are either neutrophilic and acidophilic strains, but no thermophiles have been found so far among the alkalophiles. The absolute maximal temperature for the growth of eukaryotic cells seems to be around 6 2 ° 0 and for photosynthetic algae and bacteria around 75°O a. However, such statements must be made with caution; in the case of methanogens, it was believed that all were killed above 75 oC due to a disintegration of their inner membrane systems, but since 1981 this view has had to be revised. The isolation of Methanothermus fervidus with an optimal temperature of 97°C by Stetter et al. 3 and the findings of Baross et al?'6 that bacterial communities sampled from super-heated, deepsea waters (temperatures of up to 330°C) could form methane at 100°C (and grow under high pressure at up to 300 °C) overthrew the previous notions about the temperature range of methanogens. From today's knowledge, we must expect to find the most extreme thermophiles within the group of archaebacteria. At present, biotechnologists suffer from a substantial lack of knowledge about the metabolism and natural environments of these extreme thermophiles. Their technical exploita-