5. MARCO METODOLÓGICO
6.6 LA RESPONSABILIDAD SOCIAL EMPRESARIAL DE LA VEREDA CANAVITA Y
A t e chni que t hat a l s o may b e use d t o st ab i l i z e p l asmid bor n genes to be expre s s ed in b a c t e r i a i s to have on the p l a sm i d of i n t e re s t , a gene that c ode s for a funct i on m i s s ing on the bacte r i a l host chromos ome . F o r e xample , i n se rt i ng a recomb in ant p l a sm i d with the pyrE gene i nt o E . c o l i whi ch enab l e s the s ynthe s i s o f urac i l , the reby a l l owing t he c e l l to grow in a ura c i l -f ree med i um . Another p o s s ib i l ity would be to int roduce p l a smids c oding for b a ct e r i o c i n produc t i on and immuni t y int o the s e s t r a i n s . Thi s would have two advant a ge s ; i t would enab l e t he int roduced bacter i a t o c ompete with the indigeno us b a ct e r i a in the env i r o nment , and cel l s that l o s e the p l a sm i d and t he refore immun ity t o the bacte r i o c in would be ki l le d by the bac t e r i o c i n -produc i n g c e l l s .
Another t ype of vec t o r s y s t em whi ch was oft en us ed i s lambda vect o r s . The ever i n c re as ing use of bact e r i ophage l ambda as a c l o n ing vect o r both for genom i c and cDNA has l e d t o the deve l opment of t echn i que s for t he manipulat i on of l ambdoid pha ge . The deve l opment of the phage Achromos ome a s a r ecept o r f o r f ragment s o f DNA was made by st aggeri n g c ut s within spe c if i c DNA s e quences ( t a rget s ) to produce di s c rete f r agment s with short c ohe s i ve ends ( Hedgpeth e t a I , 1 9 7 2 ; Me rt z and Davi s , 1 9 7 2 ; B i gge r et a I , 1 9 7 3 ) s uch t hat the f ragment s of DNA could be spl i ce d i nt o t he s evered a rms of a A vec t o r m o l e c ule . Two t ype s o f vec t o rs , i n se rt i o n and repl acement , have b een const ructed . A A genome with s ome n o ne s sent i a l DNA de leted, but ret a i n ing a s in g l e t arget f o r a re s t r i ct i on e n z yme , i s known a s an i n sert i on vect o r ; rep l a c ement vect ors are phage s that ret a i n t wo t a rget s f l anking a rep l aceab le segment of DNA
( Murra y , 1 9 8 3 ) .
The eff i c i e n c y of c lon i ng be i ng s uff i ci ent l
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h i gh t o a l l ow the recovery o f a c omplete l ib r a r y and rap i d h i gh dens it y s c reening t e chn i que s make l ambd a vec t o r s a very c o nvenient s y s t em t o use . Al s o t he l yt i c g rowth of l ambda rec ombi n ant s over c omes the p r oblem of secret i on , thus maki ng s c reeni n g ofCHAPTER 1 INTRODUCTION
the l ibrary e a s i e r . A l im i t at i on o f l ambda vect or s y s t ems i s t he maximum amount o f fore i gn DNA that can b e accommodate d ( approx imat e l y 2 3 kb ) a n d the di f f i cult y o f di rect s equencing from these vect ors due to the di f f i culty of preparing l arge quant i t i e s o f DNA and handl i ng o f l a rge number o f s amp l e s (Murra y , 1 9 8 3 ) . I n many cas e s , t h e capaci t y o f i n s ert i on was not rea l ly cri t i ca l . The l arge s ca l e preparat i on of DNA from a l a rge number o f s amp l e s was s o lved by a method de s cribed by Man f i o l e t t i and S chne ider ( 1 9 8 8 ) . Thi s improvement a l l ows the use of l ambda vect o r for general cl o n i n g and d i rect ly t hrough t o s e quencing w i t h o ut the nece s s it y f o r l abori o us s ubcl o n i ng steps .
1 - 4 - 2 Genet i c manipulation i n cellulase genes
The cl oning o f ce l l ulase gene s , t he determinat i on o f the ir nucl e ot ide s e quen ce s , and the anal y s i s o f the ami n o acid sequence s pre d i ct e d from t he nucl e o t i de sequence s have led to a great ly incre as e d unders tanding of s ome of the s e import ant enz ymes (Aube rt , et a l 1 9 8 8 ) . S evera l ce l l ul a s e gen e s from di f ferent microorganism have been cl oned and s t udied .
Over 2 0 appa rent l y di s t inct DNA fragment s cod i ng for ce l l ul a se have been cloned from C. thermoce l l um into E. c o l i .
(Mi l let et a I , 1 9 8 5 ; S chwarz et a I , 1 9 8 5 ; Romaniec et a I , 1 9 8 7 ) . The cl o n e s cont a in ing the gene s celA, cel B , cel C , celD and celE have been e xtens i ve l y characteri z ed and the DNA sequence s of t he coding and cont rol reg i ons determined ( B eguin et a I , 1 9 8 5 ; G repi net and Beguin , 1 9 8 6 ; Jol i ff et a I , 1 9 8 6 ; Ha l l et a I , 1 9 8 8 ) . Al s o , the x y l a n a s e gene xynZ ( Grepinet et a I , 1 9 8 8 ) and
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- g l uco s i dase gene ( Kadam and Dem�i n , 1 9 8 8 ) have bee n cl oned, cha ra cteri zed and t he DNA s equend
e o f xynZ was det e rmined .S upernat ant f rom cult ure s o f Ce o fimi cont a i ne d up t o 1 0 comp onent s with ce l l ula s e act ivity ( Langs ford et a I , 1 9 8 4 ) . The
CHAPTER 1 INTRODOCTION
c l on e s c onta in ing the genes cenA , cenB , cenC and cex have been i denti f i e d and s e quenc ed (O ' Ne i l et a I , 1 98 6 ; Wong e t a I , 1 98 6 ; Owolabi e t a I , 1 988; Moser e t a I , 1 98 9 ) .
E x tracellua r
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-glucan a s e s and x y l an a s e a re produ c e d by many members of the genus Ba ci l l us (P r i e st, 1 9 7 7 ) . Cellu la s e c l on in g i n thi s gen u s h a s h a d good p ro gre s s . The endoglu c a n a s e a n d xylanase o f B . s ubt i l i s have b e e n c loned i n E . c o l i o r o ther spe c i e s o f B a c i l l us (Nakamure e t a I , 1 987 ; MacKay e t a I , 1 986 ; Robson and Chamb l i s s , 1 987 ; K o i de e t a I , 1 98 6 ; H i n chli f f e , 1 984 ; K i m and P a c k , 1 988 ; Bern i e r Jr . e t aI , 1 983 ) . Clon ing f r om a l ka lophili c B a c i l l us sp . has a l s o been done (Fukumo r i et a I , 1 98 9 ; Kim et a I , 1 987 ; Sharma e t a I , 1 987 ; Fukumori e t a I , 1 98 6 ; S ashihara e t a I , 1 984 ) . Cloning wor k from o the r cell u l o lyti c B a ci l l us s p . was achi eved (Pa rk and Pack, 1 98 6 ; Yang e t a I , 1 988 ; S andhu and Kennedy , 1 98 6 ; P anbangred e t a I , 1 983 ; Y ang e t a I , 1 98 9 )The cellulas e gene from F . s u c c i n ogenes has been c loned (Taylor et a I , 1 987 ; G ong e t a I , 1 98 9 ; S ipat et a I , 1 987 ; I rv i n and T ea the r , 1 988 ; McGavin et a I , 1 98 9 ) . A xylanase gene f rom
F. rumi n i co l a a l s o has been c loned (Wh i tehe ad and He spe l l , 1 98 9 ) .
C l on i ng stud i e s o f c e l lula s e gen e s f rom rum i n oc o c c i a l s o h ave given e x c i ti ng re sults . The endogl u c anase gen e , exoglu c a n a s e gen e , xylan a s e gen e and
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-gluco s i da s e gene o f R. a lb u s andR. fl a ve fa c i en s h ave been c loned (Roma n i e c et a I , 1 98 9 ; H onda e t a I , 1 988 ; F l i n t et a I , 1 98 9 ; B a r r o s and Thomson , 1 987 ; Ware e t a I , 1 98 9 ; Howar d and Whi te , 1 988 ; K awa i et a I , 1 987 ; Ohm i y a e t a I , 1 988 ; Huang e t a I , 1 98 9 ) . F rom the data o f our labo r a tory (Unpubli shed data , Asmundson ) , more th
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n 1 5 p r o te i n c ompo nents have been f ound whi c h pur i fy w i th c e l l u l a s e a c tivi ty . A l s o at lea s t 8 n o n -homol ogous c l on e s have been i denti f i e dCHAPTER 1 INTRODUCTION
Oth e r ce llulas e genes have been c loned from P s e u domon a s fl u orescen s ( Le j eune e t a I , 1 9 8 6 , 1 9 8 8 ; G i lbert et a I , 1 9 8 7 , 1 9 8 8 ) ; y e a s t (Nebreda et a I , 1 9 8 6 ; Lec le rc et a I , 1 9 8 6 ) ; Erwi n i a chrys a n them i ( Brestic -Goachet e t a I , 1 9 8 9 ; Boyer et a I , 1 9 8 7 ; G i j segem et a I , 1 9 8 5 ; Kotou j an s ky et aI , 1 9 8 5 ; Aymer i c et a I , 1 9 8 8 ) ; Cryp t o c o c c u s a lbi dus (Mo r o s o li and D u r and , 1 9 8 8 ) ;
Th erm omon o spora fusca ( Hu and W i ls on , 1 9 8 8 ; L i n and W i ls o n , 1 9 8 8 ; Ghanga s and Wi lso n , 1 9 8 8 ; Collme r and Wi ls o n , 1 9 8 8 ) ; St rep t omyce s sp . ( C oppole c c h i a et a I , 1 9 8 7 ; Jaurin and Granstrom , 1 9 8 9 ; Nak a i et a I , 1 9 8 8 ) ; Ca l doce l l um s a cch a ro lyt i cum ( Love e t a I , 1 9 8 7 ) ; Agroba c t eri um sp . (Wakarchuk e t a I , 1 9 8 8 ) and r ipe avoc ado f ru i t ( Tucker e t a I , 1 9 8 7 ) .
Sever a l succe s s fu l clo n i ng studie s o f f ungi , p a rt i cula r ly T . reesei , have been reported ( Chen et a I , 1 9 8 7 ; S im et a l 1 9 8 8 ; Tee r i et a I , 1 9 8 3 , 1 9 8 7 ; Shoemak e r et aI , 1 9 8 3 ; Ars de ll et a I , 1 9 8 7 ; S a lohe imo et a I , 1 9 8 8 ) .
1 - 4 - 3 Cellulase gene structure
A gi ven m i c r oo rgan i sm wi ll produ ce a number o f ce llu las e s whi ch di f f e r i n ove ra ll amino a c i d sequence but whi ch may share short c o n s e rved s e qu e nc e s . C e llula s e s can b e grouped into fam i li e s on the bas i s of the s e conserved s equen c e s ( Tab le 1 - 3 ; Begu in , e t a l 1 9 8 9 ; Henri s sa t et a I , 1 9 8 9 ) . The loc at i on o f c o n se rved s equen c e s w i thin the enzyme s o f a f am i ly sugge sts that the fam i ly of gen e s arose by regi on o r doma i n shu f f li ng . I n b acter i a , the c on s e rved s equences o f a part i c u lar e n z yme f am i ly o c cu r in more than one genus and the c on s e rved seque n c e s o c c u r i n both Gram -pos itive a n d Gram-negative genera . Such s e qu ence conservati on i mpli e s an imp o rtant functi on for the
I
d i f f e rent e n z yme s . The s equences f o rm d i sc rete functi on a l doma ins within the e n z ymes .
The e n z yme fam i ly o f Cel l u l omon a s fimi , whi ch i n c lude s b oth an e ndoglucanase ( CenA) and an exoglu c a n a s e (Ce x ) , c onta i n two
T a b l e 1 - 3 F a m i l y A B c D F. F F a m i l i e s o f c e l l u l a s e s Org a n i sm Ce l l ul omon a s fi mi Mi crob i spora b i spora
Cl o s t r i di um t h e rmoce l 1 um 1'r i choderma reese.! Pha n a e roch a e t e ch ry s o sp o r i um C. t h e rmoce l l um C . ceJ l ul ol yt i cum C. t h e rmoce l l um Pseudomo n a s f l u o rescen s v a r . c e l l u l osa
Erwi n i . chrysan t hemi Ce o !leia R. c!J c ysan l !wm l C. a c(y t olJU t y l l cllm Ba c l l I llS sllb t i l l s al kal oph ili c Baci l l us s p . s t ra i n N - 4 s t r a i n 1 1 3 9 From : Ong e t a I , 1 98 9 . e x o g l u c a n a s e a nd e nd o g l u ca n a se A e ndog l u c a n a se A x y l a n a s e Z CHAPTER 1 INTRODUCTION ce l J o b i ohyd r o l a se I a n d I I , a nd e n d og l u c a n a s e I a n d I I I e n d o g l u c a n a s e e ndog l u c a n a se E e nd o g l u c a n a se A endog l ucanase D e n d o g l u c a n a se e n d oq l u c a n o1 se Y endoq l u c a n a se (� ndoq J u ca n a se Z endog 1 u c a f l ':l se e ndog l u c a n a se endog l u c a n a se A a l l d B e n d o g l u c a n a s e F
c o n s e rved s egment s : ( 1 ) a s equence o f about 1 0 0 amino a c ids ( at t he N - t e rm i nu s ends o f CenA but at the C - t e rminus ends o f Cex )