Genetic applications of alkaliphilic microorganisms

FEMS MicrobiologyReviews75 (1990) 279-286 Publishedby Elsevier

279

FEMSRE00145

Genetic applications ,f alkaliphilic microorganisms Koki Horikoshi Department of Bioengineering, Tokyo Institute of Technology O-okayama, Megu~-ku Tokyo. and Riken Institute Wako-ski. Saitama. Japan

Key words: Extraeelhilar proteins; Escherichia co/i; kil-gene; Secretion

I. INTRODUCTION One of the most important processes in the fermentation industry is the production of extracelhilar proteins. The reasons are as follows: (1) if gene products remain in the cells, the products cannot exceed the maximum volume of cells. However, in general, if proteins can be secreted to the culture broth, production can be increased. Also, the process of secretion allows for production from continuous culture, and substances which have an inhibitory effect on microbial metabolism may be produced as extracellular products from cells, and secretion will make the continuous production possible. (2) Usually, the number of proteins secreted from a cell is not so large, so that purification processes are relatively simple and can thus be used in fermentation industrias. Bacillus subtilis can produce enzymes in it: culture media. However, in this microorganism, host-xector systems are not yet stable enough for industrial production, and recombinant DNA products are sometimes hydrolysed by host proteases during cultivation. Another potential host for industrial production is yeast, but at present

Correspondence to: K. Horikoshi, Department of Bieengincerin8~ Tokyo Institute of Technology. O-okayama. Meguro-ku Tokyo 152.Japan.

yeast technology is not yet fully developed. Escherichia coli is widely used in genetic engineering experiments because it has been extensively studied and much is known about its genetics and biochemistry. Unfortunately. with the exception of a few proteins such as colicins [1], cloacin [2] and haemolysin [3]. E. coil does not secrete gene products from the cell. If E. coil could be modified to secrete recombinant DNA products it would be of considerable interest from the industrial point of view.

2. HOW CAN WE CHANGE E. C O L 1 INTO PERMEABLE STRAINS?. The periplasmic proteins may be secreted by the following methods: (1) Preparation of 'leaky mutants'. The outer membrane would be changed by the conventional mutation methods. However, these leaky mutants are generally not viable enough to be cultivated in large-scale fermentation vessels. (2) Secretion as a fusion product of outer mere brane protein with a periplasmic protein [4]. The outer membrane contains several proteins which have been characterized (e.g. OmpF). The outer membrane proteins are synthesized in the cells, and daring translocation into the outer membrane their signal peptides are cleaved off. Such proteins are not trapped in the periplasmic space, but

0168-6445/90/$03.50 ~o1990Federadon of European MicrobiologicalSocieties

280 EcoRI I

~ Hincll

Hlndltt / , Hlncll

---

EcoRI !

HIndlll / /Hincll

Smal - Hincll --" Hindlll

~ Sall/Hincll/

/

I

BamHI Hindlll

\

Hindll

HIndlll BamHI SalllHtncll

EcoR[

Fig. 1. Physical maps of plasmids pEApI and pEAP2. The thin lines represent the pMB9 DNA. The thick lines represent the penlcillinasegene from alkalophilic Bacillus sp. strain 170 DNA. finally found as constituents of the outer membrane. Recently, an OmpF-~-endorphin fused peptide has been secreted from E. coil Nevertheless, this method b3 very specific in that it applies only to proteins in a narrow range of molecular weight and it does not appear to have a general application. (3) Application of kil [5] or lysis.gene [2] in bacteriocins. The production of colicins and cloacin are induced by the addition of mitomycin C and the bacteriocins are exported into the culture broth. The kil.gene of ColE1 plasmid is required for the release of colicin E1 from E. coll. The H gene of cloacin DF13 plasmid plays a role in the export of cloacin [2]. The amino acid sequence encoded by the kil-gene is similar to t h o ~ of protein H encoded by cloacin DF13 plasmic and of the lysis gene of colicin E2 plasmid [2]. However, the use of antibiotics in the production of proteins is undesirable. Moreover, in the case of mitomycin C the result is cell lysis and the production of a complex mixture of intracellular and periplasmie proteins [1]. Nevertheless, under suitable conditions of promoter activation of the kil or similar genes the outer membrane may be made permeable. Recently, a penicillinase gene from alkalophilic Bacillus sp. strain 170 was cloned in E. coil by using pMB9 (Fig. 1) and it was found that the plasmid-borne penicillinase was secreted

in the culture media [6]. The dormant kil-gene in pMB9 activated by a week promoter in the inserted D N A fragment is involved in the secretion [7]. During this process of weak activation of the kil gene, periplasmic proteins such as penicillinase and alkaline phosphatases were also secreted from the cells [6]. (4) Others. Some enzymes such as one of the xylanases from the alkaliphilic Bacillus sp. No. C-125 [8,9] can be expressed in E. coli by using pBR322 and in this case the matured xylanase is released from the cells [10-13].

3, ACTIVATION O F K I L - G E N E We have succeeded in changing the outer membrane of E. coil by introducing a 2A-kb D N A fragment from the alkaliphilic Bacillus cereus. No. 170 [6], The D N A of alkaliphilic Bacillus strain No. 170, which is a penicillinase producer, was digested with EcoRl or HindIll restriction enzymes and shot gun cloned in pMB9 by conventional means. Two plasmids pEAP1 and pEAP2 were obtained from transformants. The cleavage maps of pEAP1 and pEAP2 are shown in Fig. 1. The 2,4-kb HindlI! fragment which contained the penicilfinase gene was located in the middle of the 4.5-kb EcoRI fragment. The plasmid-encoded

281 HIsc I I Eco~' t GICAACAAIA IGA/~IGTCA CAAATCTIAI AIAIATATIG T G A [ ] ] ~ CACATCA(;]5~ * i f* ) ~ mRNA ITTTTCAATG ~

IIAAGG/GTA ArO~ATCATT , CGr-tAGAI~]GT C~AI~TAI

GTTITGTTAT CAATGICv%AC~ A

180

£C~(~GTTGTAAAGTAAT~ ?GTTTGT~

~.,AAGAATC~IA /¢]GATTCCCA GTITACJd~CA I~'GATtBIGTTTGCC ,C , TAA AAGGAAI~

A~rTAI~ol LACACATOCTOCTCAGCTAGGGIAIACGGAIIGCATTTGIAGATI~;TAC~

Fig. 2. Nudcolide sequence of the Ex Ira.xment of alhalophilic #acillgs sp. strain 180, The putative -35 and -tO legions are boxed. The location and direction of the k i l gene are indicated,

The space between the H/ndlll site and the kil gene is approximately 1.4 kb in pEAP3.

penieillinase was i n m l u n o l o g i c a l l y crossed w i t h p e n i c i l h n a s e l l I o f alkaliphillc Bacillus N o . 170. P r o t e i n s i n c l u d i n g penicillinase a n d alkaline p h o s p h a t a s e usually t r a p p e d in the p e r i p l a s m i c space were released into the culture b r o t h , whereas a typical intracelhilar enzyme, fl-galactosidase, w a s n o t secreted. T h e f r a g m e n t co n tai ns the p e n i c i b linase g a n e [6] a n d a n u n k n o w n o p e n r e a d i n g f r a m e w i t h a p r o m o t e r . O u r results s h o w that the activation o f the kil-gane o f p M B 9 b y the p r o m o ter ( E x - p r o m o t e r ) in the inserted f r a g m e n t c a u s e d incrcas¢ o f the o u t e r m e m b r a n e p e r m e a b i l i t y [7]. I n s e r t i o n a l m u t a t i o n in the -35 region o f the Exp r o m o t e r i n h i b i t e d secretion. H i g h resolution S1 n u c l e a s e m a p p i n g indicated that the k i l g a n e w a s activated b y the E x - p r o m o t e r w h i c h h a d a T A T T A T sequence at -10 a n d T T G A T A at -35 (Fig. 2). T h e penicillinase w a s not r e s p o n s i b l e for this secretion.

4. S E C R E T I O N O F E N Z Y M E S E, coil car r y in g p l a s m i d p E A P 2 was aerobically g r o w n in L B - b r o t h for 20 h at 3 7 ° C a n d enz y m a t i c activities were d e t e r m i n e d . M o s t o f the penicillinase p r o d u c e d b y these cells w a s detected in the culture m e d i u m . Less t h a n 15% of the total activity w a s o b ser v ed in the p e r i p l a s m i c a n d cellular fractions. H o w ev er , a l m o s t all the fi-lactamas¢ prL~duced b y E. coil HB101 ( p B R 3 2 2 ) w a s t r a p p e d in the p e r i p l a s m i c space. E. col) HB101 c a rryi ng

01

-

. ~ ° ~

8

16

24 32r, Time (hr)

A--~--~ 40

0

48

Fig. 3. Bacterial growth and penicillinase prodtmtion by ~,"he.'qehie cui: HB10) (pEAP2). E. co?~ HBIOI (pEAP2) was inoculated in Ihe LB-broth containing 0.2~ glycerol an¢; cultured at 37°C on a rota~ shaker, Bacterial growth (absorbency at 660 nra. e) and peniciilin.~e activities (extracellular, o; intraccllular. ~; and total, A) were determined.

Table 1 DistribuUon of enzymes in /~ coli HBIOI ~ carrying plasmids Plasmids and enzymes

Activity in the foUowmg fractions (~) Extra* cellular

Pealplasmic

Cellular

None Protein b APase ~ h-gel ~

0.04 (4) 0.02 (2) 0.03 (2)

0.07 (6) 1.05 (79J 0.00 (0)

0.97 (90) 0.26 (20) 1.20 (98)

pMB9 Protein b APase '

0.01 (l) 0.0l (2)

0.03 (4) 1.32 (67)

0.75 (95) 0.15 (31)

8-gai ¢

0.00 (0)

0.00 (0)

0.80 (I00)

0.18(21) 0.29 (58) 0.09 (83) 10.70 (83)

0.12(14) 0.15 (30) 0.03 (3) 0.40 (3)

0.55(65) 0.06 (12) 0.78 (87) 1.80 (14)

pEAP2

Protein b APase c ~-gal ~ FCase d

a E. c0h strains were aerobically grown in LB-broth for 20 h at 37°C. b Protein concentration is expressed as mg in i m] of broth. Enzymatic activities of alkaline phosphatase (Apase) and fl-gaiactosidase (~-gai) are expressed as absorbance at 420 am. d Penicilfinase (PCase) ~cdvity is expressed as uni~ per ml of broth.

282

pEAP2 was inoculated in 500-ml flasks containing 100 ml of LB-broth with 0.2~ glycerol and cultured at 37°C. As shown in Fig. 3, the bacteria reached the maximum cell concentration ~t approx. 16 h, and no lysis of the cells was observed up to 48 h (viable counts 3-2 x 109). The extracellular penicillinase activity increased at about 24 h and reached the maximum at 28 h. No intracellular penieillinase was observed after 28 h cultivation. Essentially neither protein nor enzymatic activities were detected in the culture broths of E. coil HB101 or E. coli HB101 (pMB9). About two-thirds of the. alkaline phosphatase was observed in the periplasmic space and almost all of the ~-galaetosidase was in the cellular fraction. On the other hand, in E. coli HBI01 (pEAP'2), 217o of the total protein, 5870 of the alkaline phosphatase and 83~ of the penicillinase were found in the culture broth. About 87% of the /]-galactosidase activity was detected in the cellular fraction and not in the pedplasmic fraction (Table 1). These results suggest that the outer membrane of E. coil was changed by the introduction of pEAP2 into the cells, because a periplasmic enzyme, alkaline phosphatase, was released from the periplasmic space.

The pcnicillinase was not responsible for this secretion. The Ex-promoter is not very strong, almost identical to the tetracycline promoter of pMBg. The kil-gene product directly or indirectly makes the outer membrane of E. coil permeable allowing pcnicillinase to he excreted from the cells. The kil-gene and Ex-promotcr are absointely necessary for the excretion of penicillinase from the cells. The k/I gene o f ColE1 is required for mitomycin-induced lethality and the release of colicin from the cells. However, under our experimental conditions, the kil-gene did not kill the E. coil cells, but instead made the outer membrane permeable. Although the role of the kil-gene product is not clear yet, Ache [14] has succeeded in isolating the kii-gene protein from the E. coil cell envelope which had a molecular weight of 3 500. As shown in Fig. 4, the kil-gene exhibits partial homology with the gene for Braun's lipoprotein detected in the outer membraae of E. coil, not only at the level of DNA sequence, but also at the level of the amino acid sequence. Therefore, the product of the kil-gene may act as a perturbate, of the cell membrane.

I

A

111

n

+o

~su

I1 Zle&la . . . . . . . . . . .

8

5. ROLE OF K I L GENE

z~ysl,e ~ l a v a r - ~ l G n

*'"

!

so

II

II

teo

237

I ~ ~ ~ ACAJ~r.ATGeET&ETAJ~kT&C~CAAGT~ AspAJtaAlaJ~a 1a A s n G I n A ~ g L e ~ s p A s n n e t k l a ~ L y ~ T y r A ~ L y s t * *

Fi~ 4, Comparisonof nucleotideand the derivedawdnoa~idsequences of the kil 8ene (A) and tl~ lipoprot~nzene (BL Homologous regionsl, IL and Ill are boxed.

283

In case of ColE2, Pugsley and Schwartz [15] reported that the change of the outer membrane permeabifity is due at least in part to the activation of the detergent-resistant phosphofipase A by the lysis protein which closely resembles the kil gane product, although it remains unclear whether the lysis protein directly acts on the outer membrane. Aono [14] found that E. coli HB101 carrying pEAP31 which is a derivative of pEAP3 grown at 3 0 ° C released the outer membrane proteins, lipopolysaccharide and phosphatidyl ethanolamine besides penicillinase into the culture mPxlium. He analysed the contents of fatty acid in E. coil HB101 carrying or not carrying pEAP31 and no difference could be observed. Furthermore. no lysophosphatidyl ethanolamine was detected in his samples. There results suggested that the phospholipase A was not activated by our secretion vector. Recently, Suit and Luria [16] reported that expression of the kil gone of the ColE1 plasmid in E. coil kil.resistant mutants causes release of periplasmic enzymes and colicin without cell death. Phnsphnlipase A was present but not activated by kil expression in any of the mutants. Therefore, the kil peptide and lysis gone product must have different mechanisms which make the outer membrane permeable.

6. CONSTRUCTION OF AN EXCRETION VECTOR pEAP37 The pEAP3 was cleaved with EcoRI and HindIII restriction endonucleases and then trimreed by Ba131. After a fill-in reaction with E. coil DNA polymerase I Klenow fragment, the blunt ends were lisated with lisase. The ligation mixture was introduced into E. coli HB101 and the transformant which showcaJ the best excretion of pcalcillinas¢ into the culture medium waz selected. An excretion vector, pEAP31 which lacks a 1.3-kb DNA fragment between the Ex-promoter and the k// gone, was obtained. A 1.3-kb DNA fragment of the Accll digest of pBR329 contahiing the chloramphenicol acctyltransferase (CAT) gone was inserted into the Sinai site of pEAP31. As shown in Fig. 5, a ~ o m b i n a n t plasmid pEAP37 was obtained from Ap~ and Cm" trans-

T4

kil E H ~.

/" H

Oelelion~~@~H¢ E

CATfromI:~R329 5m~c Smal~1.~I- (ACCi) \

~

E He Fig. 5, Construction of pEAP37. Tile thill lille rCpteSClRSthe DNA rqIionderivedfrompMBg.The open bar represents the DNA fragmcmcontainingthe pc~icillinas¢gcnc from an alKalophili¢ Bacillus sp. and the black bar relnesente the DNA fmgnmatcontainingthechloramph~i~.ql:~ist.~aeegone(CAT) derived from pBR329. The location of the kh' gone and Ex are indicated by a black bog The dotted rims in and pEAP3indicatethe dcktion regions.

promoter pFJ~.P1

formants [17]. Most of the penicillinase activity was detected in the culture broth of E. coil carrying pEAP37. Recently, Aono [14] reported the cultivation conditions for extracelhilar production of penicilfinase on a semi-large scale. Extracellalar production of the enzyme was affected by several parameters such as concentration of carbohydrates and NaCI, pH values of culture broth, culture temperature, and aerobic conditions. He revealed that the most critical condition is culture temperature, and no secretion was observed if the temperature was lower than 26 o C. Cultivation at various temperatures (22-32°C) exhibited that the penicillinase accumulated m die periplasmic space of

284

Table 2 Effect of cultivation temperature and culture volume on the total and extraccllular production of pealcillinase

Table 3 Origin of genes used for extracellular enzyme production in E. cob

Volume (ml) 100

Name

200 300 400 500

Temperature (°C) 22 30 112.6 91.0 (1.4%) (32.0%) 153.0 93.2 (1.0%) (66.3%) 15.2 87.4 (6.6%) (71.9%) 14.6 92.0 (2.7%) (67.8%) 31.2 51.0 (3.0%) (13.3%)

37 32.8 04.0%) 48.6 (4.1%) 63.2 (39.9%) 42.2 (75,8%) 19.4 (tO.3f6)

E. co/i HBt0I (pEAP31) was inoculated into 500-ml cultivation flasks containing various volumes of LG broth and cultarod on the reciprocal shaker at various temperatures. The total and extracellular penicillinase activities wexeassayed after 36 h. The; total activity is given in units/ml. Extraceilular enz~Ine activity as a percentage of the total activity is shown in parentheses.

E. coli at 2 2 ° C was released from the cells at 3 2 ° C (Table 2). H e recommended the following conditions to produce extracellular penicillinase by E. coil HB101 carrying pEAP31. T h e organism should be inoculated in 2 0 0 - 3 0 0 ml of the L G broth (10 g Bacto tryptune (Difco), 5 g Bacto yeast extract (Difco), 2 g glycerol, 1 g glucose, and 10 g N a C I in 1 I of deionized water) in a 500-ml cultivation flask and should then be shaken at 3 0 ° C on a high-speed reciprocal shaker (172 oscill a t i o n s / r a i n with 3.2-cm strokes). Selection of cultivation conditions should be the most imp o r t a n t to produce extracellular enzymes.

pAX1

Vector used pBR322

T h r e e enzyme genes studied are listed in T a b l e 3. These genes are expressed in E. coil, but normally not secreted into the culture broth at all. Z l. Secretion vector p E A P 3 7 a n d e n z y m e genes (1) pTAX2 [17]: the plasmid pAX1 [18] was digested with B g l I ! and a 4.0-kb D N A fragment

Origin

Xylanase

Fragment Hindllt

Alkalophilic Aeromonas sp.

pAX2

pBg322

Xylanase

Bg/II

pAXI

pNKI

pBR322

Cellulase

Hindlll

Alkalophilic Bacillw N*4

pFKI

pBR322

Cellulase

Hindll]

Alkalophilic Bacillus l l ] 9

containing the xylanase gene (xyI-L) was isolated. T h i s fragment with 8 a m H I linker was inserted into the the H i n c l l site of pEAP37. F r o m the Cm-resistant, Ap-sensitive, and xylanase-producing transformants, pTAX2 was isolated. A n o t h e r plasmid, p A X 2 , was constructed by the insertion of the 4.0-kb D N A fragment with B a m H l linker into the B a m H l site o f pBR322. (2) p 7 N K 1 and p V F K l : plasmids p N K 1 [19] and p F K 1 [ 2 0 - 2 2 ] containing cellulase genes of different alkaliphilic Bacillus strains have been found in our laboratory. A 2.0-kb H i n d l l l fragment from p N K l and a 3.0 kb H i n d I l I - H i n c I l fragment from p F K I containing each eallulase genes were isolated. These fragments were filled in and ligated with HitzcIl-digested pEAP37. Plusraids p 7 N K ] and p 7 F K 1 were constructed.

Table 4 Distribution of the xylanase and cellulases in E. co?i Plasmid

7. E X T R A C E L L U L A R P R O D U C T I O N O F E N Z Y M E S F R O M E. C O L I

Gene inserted

pAX?. pTAX2 pNKI p7NK1 pFKI pTFK1 pAXI pXPI02

Extracellular fraction 23 (4.8) 135 (62.1) 12 (9.2) 282 (66.7) 7 (6.1) 93 (60.2) 10 (L6) 120(88.8)

Periplasmic fraction

147 (3o.4) 8 (3.6) 60(44.0) 92 (21.8) 88(74.3) 16 (I0.6) 290 (47.9) I0 (7,4)

Cellular fraction 315 (64.8) 74(34.3) 64(46.8) 49 (1L5) 23(19.6) 45 (29.2) 305 (50.5) 5 (3.8)

Total activity 485 217 136 423 118 154 605 135

•l'he activities (units) of the ¢azymes arc presented in each column. The percentages of the total activities arc shown in parentheses.

285 7.2. p E A P 2 and a xylanase gone (1) pXPI02 [23]: plasmid pAX1 [18] was hydrolysed with B g l l l and a 4.0-kb fragment containing the xylanase L gone was inserted in the B a m H l site of pEAP2. The plasmid pXPl02 thus constructed was obtained from Ap\ Xyl + transformants+ E. coil HBI01 carrying each of the COnstructed plasmids was cultured aerobically in Ll]-hroth at 37°C. After 24 h, the cells were harvested by centrifugation and fractionated into extracellular, periplasmic, and cellular fractions. Most of the xylanase and cellulase activities were detected in the extraecllular fractions (Table 4). In control experiments using E. coil carrying pBR322 with inserts coding for xylanase and celluiase, the enzymatic activities remained in the periplasmic and cellular fractions [17,23].

8. EXTRACELLULAR PRODUCTION OF HUMAN GROWTH HORMONE (hGH) [24] A D r a l fragment containing the promoter and the structural gone isolated from pEAP2 was digested by Rsal and connected with H i n d l i l linker. The fragment thus obtained contained the promoter and whole sequence of the signal peptide of the penicillinase. This fragment (PS fragment) was ligated with the hGH gone, inserted in the H i n d l l l site of pBR322, and introduced into E. coil The transformant (Apr, Cm') was cultured in LB-broth for 24 h at 37°C. The distribution of the hGH produced was analysed by radioimwunoassay. More than 905o of the hGH was detected in the periplasmic space of E. coll. The fragment ¢onlaining the PS fragment and hGH gone was inserted in the Hincl[ site of the secretion vector pEAP37 and introduced into E. coll. The transformant was cultured under the same conditions as described above. About 60-70% of total hGH activity was detected if, the culture broth. Western blotting indicated that the protein produced by the plasmid was immunologic.ally the same as that of the authentic hGH sample. The yield of the hGH in the culture broth was about 60-70 mg/L

9. PRODUCTION OF Fc FRAGMENT IN THE C U L T U R E BROTH [25[ The Fc fragment is a protein fraction crystallized from the papain hydrolysate of the immunoglobulin. Its molecular weight is 50000 having two disulfide bonds. The Fc has several biological activities, such as ITP (indiopathic thrum* bocytopenic purpura), protection of haemolysis etc. The DNA fragment of the Fc structural gone was ligated with a DNA fragment endocing the pen~illinase signal peptidc isolated from alkaliphilic Bacillus No. 170 and inserted in a S a i l site of the secretion vector pEAP37. After introduction of the plasmid into E. coli HBIOI and cultivation in LB-broth for 24 h, more than 605O of the gone products were detected in the culture broth. Without the signal sequence, no gone product was detected either in the periplasnfic space or the culture broth. In conclusion, we believe that the secretion vector pEAP37 may prove 1o be useful industrially for producing extracellulur proteins from genetically modified E. coll.

REFERENCES: [l] Zhan8, S., Faro, A. and Zobay. G. 11985)Mitomycin.induccd lethality of Escherichia coli cells containing the ColEl plasmid; :nvolvemcntof the kil gone. J. Bactenol 163, 174-179. [2] De Graaf. F.K. and Oude$a+ B. (19~6) Production and release of cloacin DFI3 and relaled ¢.olicins.in Current Topics in Microbiologyand ImmunologS., Vol. 125 (Wu. H.C. and Tai, P.C., Eds.). pp. 183-205. Springel'oVerla$, Berlin, Heidelberg,New York, Tokyo. [3] Mackman+N., Nicaud, J.-M., Gray, L. and Holland, I.B. (1986) Secretion of haemolysin by Escher~chia coli, in Current Topics in Microbiologyand Immunology,Vol. 125. (Wu. H.C. and Tai, P.C., Eds.), pp. 159-181. Springer-'~'cztag, Berlin.Heidelberg,New York.Tokyo. [4J Nagahan. K.. Kanayarna, S.. Manaka~, K.. Aoyagi, y. and Mizushima, S. 11985) Secretion in[o the culture mediumof a foreigngcne product from Escherichia cob: use of the ompF gone for secretion of human beta-endotphin. EMBOJ. 4. 3589-3592. 15] Chan. P.T., Ohmori. H,, Tomizawa, J. and Lebowil~J+ 11985) Nuc]eotide Sl~luenct:and gone ofga~zalion of CoIEI DNA. J. Biol.Chem.260. 8925-8935. [6] Kudo.T.. Kalo. C. and Horikoshi,K. (1983) Excretionof the penJcillinaseof Ihe alka|iphilicBocillus sp. Through the Escherlchia coil outer membrane. J. Bacteriol. 156, 949-951.

286 [7] Koheyashi, T., Ka¢o, C., Kudo, T. and Horikoshi, K, (191~6) Excn:lion of Ihe penici]linase of an alkaliphifie BaciJius sp. through the £scherichia coli outer membrane is caused by insenional activation of the kll gene in plasmJd pMB9. J, Bacteriol. 166, 728-732. [8] Honda, H., Kudo, T., Ikura, Y. and Horlkoshi, K. (1985) Two types of xylanases of alkaiopbJlic Bacill~ sp. No, C-125. Can. J. Mierobiol. 31,538-5d2. [9] Honda. H. Kodo. T, and Horikoshl. K. (1985) Purification and partial cheracte~zation of alkaline xylanase from Escherlchia coil carrying pCX311. Agricol. Biol. Chem. 49. 3165-3169. [10] Honda. H., Kudo, T. and Horikoshi, K. (1985) Molecular cloning and expression of xylanase gene of alkaliphific Bacillus sp. strain C-125 in Escherichia coll. J. Bacteriol. 161, '/84-785. [11] Honda, H., Kodo, 1". and Horikoshi, K. (1985) Purification and partial characterization of alkaline xylanase from Escherichia coli carrying pCX311. Agricol. Biol. Chem. 49, 3165-3169. [12] Honda, H., Ku¢~o. T. and Hotikoshi, K. (1985) Selective excrelion of alkaline xylanase by Escherichia coil carrying pCX311. Agricol. Binl. Chem. 49, 3011-3015. [13] Honda, H., KOdo, T. and Horlkoshl, K. (1986) Extracdlular production of alkaline xylanase of alkalophilic Bacillus sp. by Escherichia coil carrying pCX31L J. Ferment. TechnoL 64, 373-377. [14] Aono, R. (1988) Cultivation conditions for extracellular production of penicillinase by Escherichia coil carrying pEAP31 on a semi-large scale. Appl. Micxobio|. BiotcchnoL 28, 414-418. [15] Pugsley, A.P. and Schwartz, M. (1984) Colicin E2 release: lys~s, leakage on secretion? Possible role of a phosphulipaso. EMBO J. 3, 2393-2397. [16] Suit, J.L. and Luria, S.E. (1988) Expression of the kit liene of the colEl plasmid in Escherichia co// kil resistant mutants causes release of pefiplasmic enzymes and of colicin without cell death. J. BaeterioL 170, 4963-4966.

[17] Kato, C., Kobayashi, T., Kudo, I". and Horikoshi, K. (1986) Construction of an excretion vector: extracellular production of Aeromonas xylanase and Bacillus cellulases by Eschertchta coOl. FEMS MicrobioL Lett. 36, 31-34. [18] Kudo, T. Ohkoshi, A, and Horikoshi, K. (1985) Molecular cloning and expression of xylanase gent of alkalophillc Aermnonas sp. No. 212 in Escherichia coll. J. Gen. Microbiol. 131, 3317-3324. [19] Sashihara, N., Kuda, T. and Horikoshio K. (19~4) Molecular cloning and expression of cellulase genes of alkalophilic Bacillus sp. strain N-4 in Escherichia coll. J. Baeterinl. 158, 503-506, [20] Hotikoshi, K., Nakao, M., Kurono, Y. and Sashihara, N. (1984) Celiulases of an alkaliphilic Bacillus strain isolated from soil. Can. J. Microbiol. 30, 774-779. [21] Fukumori, F., Kudo, T. and Horikoshi, K. (1985) Purification and properties of a cellulase from alkalophilic Bacillus sp. J. Gen. MicrobioL 131, 3339-3345. [22] Fukumorl, F., Sashihara, N., Kudo, T. and Itorlkoshi, K. (1986) Nucleolide sequences of two celitllase genes from alkaliphi[ic Bacilltu sp. strain N-4 and th¢ll strong homology. J. Bacterin]. 168, 479-485. [23] Kato, C., Ohkoshi, A., Kudo, T. and Horikoshi, K. (1986) Exlrac~llular production of xylanase L in F,scher~chia coil using excretion vector pEAP2. Agricol. Biol. Chem. 50, 1067-1068. [24] Kato, C., Kobayashi, T., Kudo, T., Furuksato, T., Mutakami, Y., Tanaka, T., Baba, H., O i L , T., Olsuka, E., Ikehara, M., Yanngida, T., Kato, H., Moriyama, S. and Hofikoshi, K. (1987) Construction of an e~cretinn vector and extraceliulat production of human growth hormone from Escherichla coll. Gene 54,197-202. [25] IOtal, K., Kudo, 2"., Naka, nura, S.. Maegi, T., ]chkawa, Y. and Horikoshi, K. (1998) Extracellular production of human immunoginbulin G Fc region by E~herichia coll. Appl. Microbiol. BiotechnoL 28, 52-56.