Lee Et Al-1997-Biotechnology Progress

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Sequential δ-Integration for the Regulated I nsertion of ClonedGenes in S a c c h a r o m y c es c e r ev i si a e

Frank W. F. Lee† and Nancy A. Da Silva*

Department of Chemical and Biochemical Engineering, University of California, Irvine, California 92697-2575

A novel δ-integra tion vector w a s developed to a llow the sequent ial insert ion of multiplecloned genes in the yeast Saccha r omyces cer evi siae . To allow repetitive integrat ions,the reusable U R A 3 B last er selection casset te w a s employed; the insert ions (of CUP1p- lacZ in this study) were selected using the U R A 3 ma r ker w h i ch w a s s u b s eq u en tl y“ pop ped” ou t b y r ecomb in a t i on b etw een f la n ki n g d i r ect r epea ts . Tr a n s f or ma n t scon ta i n ed on l y o n e n ew i n tegr a t ed c op y a f ter th e l os s o f th e U R A 3 m a r k e r , a n dsubsequent tra nsforma tions were effective for the sequential insertion of a series ofgenes (one at a time) into dispersed chromosomal δ sequences. The structura l stabilityof th e integrat ions wa s location-dependent (ra nging from 75% to 100% aft er 50generat ions in complex medium w ith or w ithout gene expression), a nd t he integra tions(at least up t o f ive) ha d no significan t effects on t he growth of the cells. In a ddition, -gala ctosidase specific a ctivity levels va ried linearly w ith integrat ed copy n umber.The repetitive, regulated nature of integration with this vector is not possible withtraditional δ-integration or other homologous recombination methods, and is promising

for f ine-tuning cloned gene copy number a nd for the insertion of metabolic pat hwa ygenes.

Introduction

The yea st Saccharomyces cerevisiae is a very importa ntindustr ial microorganism a nd plays a key role in indus-tries ranging from pharmaceutical production to ethanolan d biomass production. In a ll of these applica tions, theab il i ty to introduce stab le foreign genes is extremelyimporta nt, a nd t he ab il i ty to engineer t he metab olism oft h i s y ea s t h a s t r em en d ou s p ot e n t ia l . S. cerevisiae ,however, has a very limited range of plasmid vectors; only

tw o are generally useful : CE N -b ased plasmids a t one totw o copies per cell an d 2 µ-based plasmids at 10-50 copiesper cell (or up to 100 copies with the leu2-d selectiong e ne ) (E r h a r t a n d H o ll en b er g , 1 98 3; R om a n o s e t a l . ,1992). Fine-tun ing of copy num ber is not possible, yetoptimizing t he num ber of cloned genes can be extremelyimporta nt f or cells synt hesizing toxic compounds orsecreting complex proteins. Furt hermore, for the intro-duction of metab olic pat hwa ys into yeast , plasm ids mayb e t o t a l ly u n su it a b l e. I n g en er a l , o n ly on e cl a s s ofplasm ids can b e stab ly ma inta ined in the cell at a t ime,and only one or tw o pat hwa y genes ca n b e carr ied on theplasmid. However, several pathw ay genes must often b eintroduced, ideally with precisely regulated expression.Directed integrat ion of the genes into the host chromo-

somes is an ideal method for introducing these genes;cloned gene loss during cell division is eliminated and,for st ructurally st ab le insertions, t he optimum numb erof genes can b e maintained constant.

Trad itional integrat ion methods a re l imited in t ermsof t h e n u m be r of g en es w h i ch ca n b e i n s er t e d, t h estab il i ty of the integrated genes, and the regulation ofthe integration process. Homologous recombinat ion isthe standard method for introducing cloned genes intot h e y e a s t g en om e . G e n e r a l Y I p p la s m i d s t y p i ca l l y u s e

auxotrophic markers (e.g., U R A 3 a n d T R P1 ) a s t a r g etsites a nd a re integra ted a t only one to tw o copies per cell(Romanos et al., 1992). To insert additional copies of thesame (or dif ferent) genes, a new ma rker and t arget si temust be used. To obta in more copies of the cloned genein one round of tra nsformat ion, th ese auxotrophic mark-ers have been replaced by repetitive chromosomal DNAsequences (e.g. , the r ib osomal DNA cluster and δ se-quences) (Lopes et a l. , 1989; Sa kai, 1990, 1991). U singdomina nt selection mar kers, more tha n 40 copies can be

integra ted a t one time; however, these integra tions occurprimarily in a t and em arra y (Pa rekh et al . , 1996; Shiomiet a l . , 1995; Wan g et al . , 1996). Tan demly repeat edcopies have a higher probability of being looped out byexcisiona l recombina tion (Roma nos et a l. , 1992), a nd thesta b ili ty of the insertions must b e carefully determined(Fujii et al . , 1990; Wang et al . , 1996). Work in ourl a b or a t o r y h a s d e m on s t r a t e d s i gn i fi ca n t s t r u ct u r a l i n -sta b ili ty w hen expression cassett es (M F R 1P /L-S U C 2 a n dCUP1p-lacZ ) ar e integrated in this ma nner (Lee and DaSilva, 1997; Wang et al., 1996). These current homologousr e com b in a t i on m e t h od s a l s o d o n o t a l l ow r e gu l a t e d ,sequentia l cloned gene insertions. Auxotrophic ma rkersare quickly depleted, and when antib iotic selection ise m pl oy e d , o n ly a s i n gl e r o u n d o f t r a n s f or m a t i o n i su s u a l ly p os s ib le . O n ce o n e o r t w o g e n es h a v e b ee ninserted, the selection of super-resistant strains occursat a much higher frequency than subsequent transforma-tions (unpublished data).

The goal of our work was to develop a new homologousrecomb ination method which results in dispersed (notta ndem) integra tions and w hich can be used repetitively.This would improve the structural stability of the inser-tions and al low the sequentia l integration of a series ofthe sa me or different genes. To accomplish this, w e havecombined a reusable selection marker (the U R A 3 B l a s t e rcassette) with t he δ ta rget seq uence. There a re ca. 425 δsequences dispersed t hroughout the yeast genome (D u-jon, 1996), an d each is a potent ial t a rget sit e for integra -

† Current a ddress: Department of Chemical Engineering, Uni-versity of Colorado, Boulder, CO 80309-0424.

* To whom all correspondence should be addressed. P hone:(714) 824-8288. F AX: (714) 824-2541.

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S8756-7938(97)00055-6 CCC: $14.00 © 1997 American Chemical Society and American Institute of Chemical Engineers


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tion. The U R A 3 blast er cassette w as originally developedfor t he construction of multiply disrupted yeast stra ins(Alani et al., 1987) and allows the repeated used of U R A 3

selection. The cassette consists of a yeast U R A 3 genef la n k e d b y t w o d i r ect b a c t er i a l hisG re pe a t s . Af t erselection for the U R A 3 gene on selective plat es, this genecan be lost by excisional recombination between the twohisG r ep ea t s , a n d ce ll s w h i ch h a v e l os t t h e U R A 3 gene(s) can be selected on 5-FOA (Boeke et al. , 1984).We have incorporated the U R A 3 B l a s t e r c a s s e t t e a s t h es el ec t ion m a r k e r on ou r δ-integrat ion vector (δ/U B -i n t e gr a t i o n ) a n d h a v e d e m on s t r a t e d t h e a b il it y t o p e r -f or m s e q u en t i a l c lon e d g e n e i n t e gr a t i on s of p r ec is enumbers of genes into dispersed chromosomal sites. Themodel gene inserted was the Escheri chia coli lacZ geneunder t he cont rol of the indu cible yeast C U P 1 promoter.Th e s t r u ct u r a l s t a b il it y of t h e i n t eg r a t i on s a n d t h erelationship b etween integrat ed cloned gene numb er,growth, a nd expression w ere determined.

Materials and Methods

Strains. Haploid S. cerevisiae strain TMy16 (M A T at r p 1 -∆901 u r a 3 -5 2 h i s 3 -∆201 ade2 -101 l y s2 -1 l e u 1 -12 can1 -100 G AL +) (Kirchner and Sandmeyer, 1993) wasemployed for the integration experiments. E. col i XL1-B l u e (F ′ [proA+ proB + lacI q lacZ ∆M 15 Tn10 (T et R)]/supE 44 hsdR 17 r ecA1 gyrA 46 t hi r el A 1 l ac -) (Stra ta gene,CA) wa s used for plasmid constr uction a nd ma intena nce.

Plasmids. P lasmid pBluescript II S K(-) (Stra ta gene,CA) served as the b ackb one of the δ-integration vector.P las mid pH 3 (B oeke et a l., 1985) is a CE N -based plasmidcontaining a native Ty1 retrotransposon (Ty1-H3) with

a unique X h o I restriction site wit hin the 5′ en d δ element .

P lasmid pLD3 (Durrin et a l. , 1991) carries a CUP1p-lacZ cassette. Plasmid pNKY51 (Alani et al. , 1987) containst h e U R A 3 B l a s t e r c a s s et t e .

Construction of Vector pδ-UB. The U R A 3 B l a s t e rcassett e wa s isola ted from pla smid pNKY51 (Alani et a l. ,1987) by B am H I a n d B gl II rest riction digests. This 3.8kb B am H I /B gl I I f ra g m e nt w a s t h en i n se rt e d i n t o t h eB am HI si te with in th e multiple cloning si te of plasmidpBluescript II S K (-). To ensure different t ra nscriptiondirections f or the U R A 3 B l a s t e r a n d CUP1p-lacZ cas-s et t e s i n t h e f in a l con s t r uct , t h e or ie nt a t i on of t h efragment shown in Figure 1a w as confirmed b y B am H Ia nd E co RI restr iction digests. Sub sequently, an 840 b pPv u I I /H i n d III f ra gment conta ining the 5′ δ of native Ty1

from plasmid pH3 (B oeke et a l . , 1985) wa s t reat ed withT4 D N A p ol ym er a s e t o f il l i n t h e 5′-o ve r h a n g a n dinserted into the X h o I si te within the multiple cloning

site (the 5′-overhang of the X h o I si te w as also f i lled in).An or i en t a t i on op pos i t e t o t h a t of t h e U R A 3 B l a s t e rcassette was confirmed b y B am H I a n d X h o I restrictiondigests. The f inal vector wa s designated pδ-UB (Figure1a) an d conta ins the δ sequ ence, U R A 3 Blaster cassette,a n d u n iq u e Sa c I , B st XI , Sa c I I , N ot I , X ba I , B am H I ,E co RI , S al I , a n d K pn I s ites for cloned gene insert ion. Theunique X h o I s it e w i th in t h e δ s e q u en ce i s u s ed f orl inear ization of the plasm id b efore tr an sforma tion.

Media. C omplex YP D medium (Sherma n et a l. , 1986)w a s u s e d f or t h e g r ow t h of S. cerevisiae . S D C a nd SD -5-FOA plates were prepared as described previously (Leeand Da Silva, 1996a). LB medium (Sambrook et al., 1989)w a s t h e E. c ol i nut rient medium; 50 mg/L a mpicillins o d i u m s a l t ( S i g m a ) w a s a d d e d t o s e l e c t f o r p l a s m i d -containing (A m p R) cells. LB-X-gal plates (Sambrook eta l., 1989) conta ining 40 mg/L X-ga l a nd 50 mg/L a mpi-cillin sodium sa lt w ere used t o check for -galactosidaseactivi ty in E . col i .

Cultivation. All seed tub e cultures and b atch cul-tures for the a ctivi ty a ssays were conducted in 16× 125m m c u lt u r e t u b es c on t a i n in g 5 m L of m e d iu m . B a t c hcu l t u r es f or t h e s t a b i l it y a n d g r ow t h r a t e e xp er i m en t swere conducted in 250 mL f lasks containing 20 mL ofmedium. All tub es and f lasks were incub ated in an a irshaker (New Brunswick, model G25) at 30 °C and 250r p m . B i o m a s s c on ce n t r a t i on s w e r e d e t e r m in e d f r omoptical density measurements at 600 nm (NOVASPECII spectrophotometer , P har ma cia ).

DNA Manipulation and Yeast Transformation.Lar ge-scale plasmid DNA preparat ion from E. col i w a scarried out using the Qiagen Plasmid Maxi kit (Qiagen).Tra nsforma tion of yeast wa s carried out using the lithiumacetat e method (Ito et a l . , 1983). Isolation of tota l DNAfrom yeast was performed according to the proceduredescribed by B oeke et a l. (1985).

Southern Hybridization and -Galactosidase As-says. The lacZ probe wa s a 3.3 kb B am H I /Sa l I f ragmentof plasmid pLD3 (Durrin et al. , 1991). The hisG probew a s a 1 . 1 k b B gl I I /St u I f ragment of plasmid pNKY51(Ala ni et al., 1987). Nonra dioact ive colorometr ic hybr id-ization and detection wa s performed using the GE NIU Skit (Boehringer M annh eim). -G a lactosida se specificactivi ty assa ys were conducted as fol lowed. Cells were

Figure 1. P l a sm i d m ap s of (a) pδ-UB and (b) pδ-la c Z- UB showing relative size, restriction sites, and location of inserted DNA.

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grown at 30 °C for 6 h with copper sulfate (1 mM) addedto the culture for the la st 30 min of incubation. Sa mpleswere then permeabilized with isopropyl alcohol (Sriencet al . , 1983). -G a l a c t os i da s e s p eci fi c a c t i vi t y i n t h e

cultur e (Abs420/min/g of cells) wa s mea sured via a n ONP Gassay on the permeabilized samples (Da Silva and Bailey1989) a t 420 nm (DMS 100S U V-vis spectrophotometer,Varian).

Results

Construction of Sequential δ/UB-Integration Sys-tem. A δ-integration vector, pδ-UB (Figure 1a), contain-ing the δ ta rget si te, the U R A 3 Bla ster selection cass ette,a n d u n i q u e r e s t r i c t i o n s i t e s (Sa c I , B st XI , Sa c I I , N ot I ,X ba I , B am H I , E co RI , Sa l I , a n d K pn I) for cloned geneinsertion w as constructed. The deta i ls are provided int h e M a t e r ia l s a n d M et h od s . Th e 3 .6 k b E co RI /X h o Ifragment conta ining the CUP1p-lacZ expression casset te

from plasmid pLD3 (Durrin et al., 1991) was inserted intot h e u n i q u e E co R I a n d Sa l I s it es on pδ-U B t o f or mp la s m i d pδ-lacZ -U B (F i g u re 1 b ). I n t h i s v e ct o r , t h eorienta tion of the CUP1p-lacZ expression cassette (whichlacks a terminat or) is the same as t ha t of the δ element ,and th e terminat or within the δ element is utilized. TheU R A 3 B l a s t e r a n d CUP1p-lacZ cassett es ha ve oppositetra nscription directions. In plasmids pδ-UB and pδ-lacZ -U B , t he X h o I s i t e w i t h i n t h e δ element is unique for thelineariza tion of the plasmid b efore tra nsf orma tion.

Verification of U R A 3 Blaster Cassette Function.P l a s m i d pδ-lacZ -UB was linearized by restriction digestw i t h X h o I a n d u s e d t o t r a n s f o r m S. cerevisiae s t r a i nTMy16 (Figure 2a,b). Cells wit h int egra tion(s) (Figure2c) were selected on ura cil-def icient SDC plates. The

t r a n s f o r m a n t s w e r e c u lt u r e d i n t u b e s c on t a i n in g n on -s el ect i v e Y P D m e d iu m a n d t h e n s p r ea d on t o 5 -F O A-conta ining plates t o select cells wh ich had lost the U R A 3 gene by excisiona l recombina tion betw een the t wo directb acterial hisG repea ts (Alani et a l., 1987). The fina l cellswere grown on uracil-deficient S DC plates t o confirm t heloss of the U R A 3 gene.

C e ll s a m p le s b ef or e a n d a f t e r U R A 3 c ur i n g w e r ea n a l y ze d b y S ou t h e r n h y b r id i za t i o n s . G e n om i c D N Asa mples were isolat ed from these integrant s an d digestedby restriction endonuclease E co R I ( a s s h o w n i n F i g u r e2 ). Th e p r ob e w a s a 3 .3 k b lacZ fr a g m e n t . A s a m p l eSouth ern blot is shown in Figure 3a. Typically, only oneor two (tandem) b ands were ob tained af ter transforma-tion. The ba nd a t ca . 11.27 kb (the size of the integra ting

vector) in lane A indicat es the E co R I f r a g m e n t s a r i s in gfrom t he inner sequence within t he ta ndem copies. Theo t h e r b a n d r e p r e s e n t s t h e f r a g m e n t a t o n e e n d o f t h e

ta ndem repeat . Regardless of the numb er of copies atan integration si te b efore U R A 3 curing, only one copyremained at that location after the excisional recombina-tion b etw een t he tw o direct b acterial hisG repeat s. Thisi s l i ke ly b ec a u s e a n y t w o hisG repeats can recombine,and we have selected for the recombination event whichexcises all U R A 3 genes (due to 5-FOA counter selection).A sa mple stra in (before an d a fter U R A 3 curing) carry inga s i n gl e i n t eg r a t i on w a s a l s o h y br i d iz ed w i t h t h e hisG prob e (Figure 3b ). As predicted, t he size of the b anddecreased a fter excision of the U R A 3 gene. This decrea sew a s 2 . 7 k b, t h e l en g t h o f t h e U R A 3 g e n e a n d on e hisG repeat ; t herefore, t he U R A 3 B laster cassette perf ormedas expected.

Recombinat ion between t he flanking δ sequences (rather

t h a n t h e hisG repeats) can also occur during the 5-FOAselection step and r esult in loss of the entire insert. Thefrequency of this event varied for dif ferent integrationloca tions (ra nging from 0%to 80%) but w a s often below 10%. Therefore, in our work, 20 sa mples w ere routin elyassayed b y Southern b lot af ter U R A 3 curing. We foundthat a high level of instab il i ty (of the entire insert) atthis 5-FOA step indicated a generally unsta b le integra -tion location.

Identification of Sequential Integrations. Thegoal of employing the U R A 3 Blaster selection cassettew a s t o a l low s eq u e n t ia l , d i s pe r se d i n t e g r a t i on s of t h esame or dif ferent genes via δ-integrat ion. To test thesuccess of this method, the integra nts (af t er U R A 3 loss)were retra nsformed with the original int egrating vector

Figure2. ( a) P l asm i d pδ-lacZ -UB after l inearization with X h o I .The δ vector (b) before and (c) after homologous recombinationi n t o t h e c h r om osom al t a r g e t se q u e n ce . Th e f i l le d a r r ows an dthick lines represent the vector-borne elements. The open arrowsand thin l ines represent the chromosomal sequences.

Figure 3. (a) Southern blot of stra in TMy16 after tra nsforma-tion/integr a tion wit h linear pδ-lacZ -UB . Genomic DNA wa s cutw i t h E co R I a n d h y b ri di ze d w i t h t h e lacZ probe. Lanes A, C,an d E ar e t h e st r a i n s b ef or e U R A 3 curing. Lane A contains t wot an d e m c op i e s; l an e s C an d E ar e si n g l e c op i e s. L an e s B , D ,a n d F a r e t h e r e s u lt i n g s t r a i n s f r om l a n es A , C , a n d E a f t e rU R A 3 curing. Lan e M is t he 1 kb ladder DNA mar ker (G ibco).(b) Southern blot of a TMy16 tra nsformant with one integratedcopy of plasm id pδ-lacZ -UB . Genomic DNA was cut w ith E co R Ian d h y b r i d i z e d se p ar at e l y wi t h t h e h is G ( l an e s A an d B ) an dlacZ ( lanes C and D) probes. Lanes A and C are the same DNAsam ple before U R A 3 c u r i n g . L an e s B an d D ar e t h e r e su l t i n gstrain (from lanes A and C) af ter excision of the U R A 3 gene.

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and t he procedure wa s repeated. The new integrationsca n occu r a t a n ew l oca t i on or i n t a n d em w i t h t h eprevious integration; b oth have b een ob served in thisw o r k. H o w e ve r , on l y t h e n on t a n d e m i n t eg r a t i o ns r e -ma ined a fter plat ing on 5-FOA (a nd excisional loss of theU R A 3 gene). This is similar to the b ehavior af ter t hef irst round of tra nsforma tion. Therefore, only a n inte-grat ion ta rgeted to a new locat ion increases the final copynumber. After each round of tra nsformat ion, a Southernhyb ridization was performed to select cells with new,nonta ndem insert ions. The procedure can be performed

repeti t ively t o increase integrat ed copy numb er b y onegene at a t ime. To da te, five CU P1p-lacZ cassettes haveb een sequential ly inserted into dispersed chromosoma ll oc a t i on s ; t h e i n t e g r a t i on p a t t e r n s of t h e se s t r a i n s a r es h ow n i n F i g u r e 4. E v e r y b a n d d i ff er s i n s i z e (a n d i snot 11.27 kb); therefore, the nontandem single integra-tions have occurred at f ive distinct locations.

Characterization of Integrated GeneE xpression.For the resulting st ra ins to be useful, subsequent expres-sion and control of the δ-integrated genes is essential .To s t u d y t h e e ff ect of cop y n u m b er (a n d i n t eg r a t i o nlocation) on expression, -galactosidase specific activitywa s a ssayed f or the st ra ins carrying one to f ive copies oft h e i n t e g r a t e d CUP1p-lacZ cassett es (Figure 4). Theuninduced ba ckground a ctivity wa s less tha n 10%of tha tu n d er i n d u ce d con d i t ion s f or e ve r y s t r a i n (d a t a n otshown); therefore, the C U P 1 promoter is well regulated.The inducer was 1 mM CuSO4, a n d t h e i n d u ct i on t i m ewa s 30 min. The results (Figure 5) clearly demonstrat et h e l in e a r c or r e la t i on b et w e e n s p ec if ic a c t i v it y a n di n t eg r a t e d cop y n u m b e r . Th e -galactosidase specificactivi t ies associated with single inserted copies of thelacZ gene w ere also equivalent to t ha t f or one copy of aCE N -b ased plasmid carrying the CUP1p-lacZ cassette(Lee a nd D a Silva, 1996b ). Therefore, insertion into δsequences ha s n o negat ive ef fect on expression. The δsequences t hus provide excellent t a rgets for t he insertionof expression cassettes.

Growth rate studies were performed in YPD medium

w i t h a n d w i t h o u t 1 m M C u S O4 induction for th e stra inscarrying one to five dispersed δ-integrated CUP1p-lacZ cassettes. Growt h rat es varied b y less tha n 6%(higheror l ow e r ) com p a r e d w i t h t h e or i g in a l h os t (d a t a n otshown). Therefore, the dispersed δ-mediated integra-tions (at least to a copy numb er of 5) had no signif ican teffect on growth even with expression of the cloned gene.

Structural Stability of Dispersed δ-Mediated In-tegrations. The structural stability of δ/U B -int egra tionswa s a na lyzed f or the str ain carrying f ive copies (Figure4, lan e E) with a nd with out lacZ gene expression. Eigh tsequential batch cultivations (with at least seven genera-t i on s p er cu l t u r e) w e r e p er f or m e d i n co mp le x Y P Dmedium (with a nd w ithout 1 mM CuSO 4), a sample wasspread on YPD plates, a nd 30 colonies from ea ch set were

randomly selected for the structural stab il i ty analysis.The -ga lactosidase specific activity of ea ch str a in (colony)w a s t h en a s s a y ed v ia O NP G t es t s, a n d t h e s a m pl essepara ted into two groups. Group I retained the originala ctivity level (before long-term cult ure), a nd G roup II ha d

reduced activity. Southern blots were performed on tw o-thirds of the samples in Group I and al l of the samplesin Group II . All assayed sa mples from G roup I reta inedthe f ive integra ted CUP1p-lacZ cassettes. The samplesi n G r o u p I I , h o w e v e r , h a d l o s t o n e o r t w o i n t e g r a t e dcopies. The results a re shown in Ta ble 1.

The average copy numbers in the final population were4.70 and 4.57 (Table 1) after more than 50 generationsin complex medium without a nd w ith lacZ gene expres-sion, respectively. Therefore, cloned gene expression didnot signif icant ly af fect structura l stab il i ty. I t should b enoted tha t in a l l cases t he same t wo (of f ive) b ands w erelost ; the other three b ands were alw ay s present, indicat -i n g e x t r em e ly s t a b l e i n t e gr a t i o n s. Th i s s u g g es t s t h a tstructural stab il i ty of δ-mediated integrat ions depends

on th e specific δ ta rget site utilized. The strain reta iningthe t hree sta b le copies could b e used for further t ra ns-forma tions to obta in insertions a t only very st ab le loca-tions.

Results of “Blind”Sequential Integrations. Therear e ca. 425 δ elements in th e yea st genome (Dujon, 1996),an d each is a potentia l target for integration. However,as discussed ab ove, repeat ed tra nsforma tions can resulti n n e w i n t eg r a t i on s a t a l r e a d y oc cu p ie d l o ca t i on s a n dt h u s t a n d e m r e pe a t s . Af t er U R A 3 curing, these new integrant s did not ha ve higher copy numb er. Furth er-more, 5-FOA counterselection may result in loss of thee n t ir e i n t e g r a t i n g p l a s m i d v i a r e com b in a t i on a t t h e δs eq u en ce s r a t h e r t h a n t h e hisG r e pe a t s . H o w e ve r ,performing S outhern hyb ridizat ions af ter each t ran sf or-

ma tion to avoid selecting the t an dem insertions, or a f ter5-FOA selection to avoid loss of the entire insert, is time-consumin g. Therefore, the success of “blind” sequentia lintegrations (without Southern a na lysis) wa s evaluated.

S t r a i n T M y 1 6 w a s t r a n s f o r m e d w i t h l i n e a r pδ-lacZ -UB, and 10 single colonies were picked from the SDCtransformation plates, mixed and cultured together innonselective YP D m edium, an d t ra nsferred to S D-5-FOAplates. Ten single colonies were selected f rom theseplates, mixed, and used for the next transformation; thiscycle was repeated severa l t imes. No Southern hyb rid-izat ions were performed between rounds. The distribu-tion of integrated copy numbers and integration patternswa s determ ined via S outhern blots on 20 single coloniesa f t e r f i v e a n d 1 0 r ou n d s o f t r a n s f or m a t i on (d a t a n ot

Figure 4. Southern blot demonstrating sequential integrationof the CUP1p-lacZ cassette via δ/UB -integra tion. Stra ins withone to f ive sequentially inserted copies of the cassette (af terU R A 3 c u r i n g ) ar e sh own . G e n om i c D N A was c u t wi t h E co R Ian d h y b r i d i z ed wi t h t h e lacZ probe. Lane M is the 1 kb ladderDNA marker (Gibco).

Figure 5. -Galactosidase specific activity versus copy numberof the CUP1p-lacZ cassett e inserted into host st ra in TMy16 viaδ/U B -i n t e gr at i on . D a t a wi t h e r r or b a r s r e p r ese n t a n a ve r ag eof multiple three to six strains w ith th e same copy number. Dat awi t h ou t e r r or b ar s a r e t h e r e su lt s f or a s i n g le st r ai n .



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shown). Only six integration patt erns existed af ter f iverounds, and only thr ee after 10 rounds. After five roundsof tra nsforma tion a nd selection, the percenta ge of colo-nies w ith one, tw o, an d t hree insert ions were 15%, 70%,a nd 15%, respectively. After 10 rounds, t he percenta geof colonies with tw o and three insertions w ere 25%a nd75%, respectively (none h a d only one insert ion). Themaximum integrated copy numb er ob tained in a singlecell wa s 3 after both five and 10 rounds of tra nsformat iona n d s el ect i on . Th i s i n d ica t e s t h a t m a n y r ou n d s o fretran sforma tion were unproductive. The new integra-tions were in a ta ndem arr ay with the previous ones, ort h e e n t ir e i n s e r t w a s l os t d u r in g t h e cu r i n g p r oce s s;

therefore, for several rounds, no increase in copy numberoccurred after U R A 3 curing (see previous discussion).Southern hybridizations between each round eliminatedthis problem.

Discussion

A novel integration vector employing a reusable (U R A 3 Bla ster) selection cassette a nd a repeti t ive target si te (δ)ha s been developed for regulat ed cloned gene insert ionsinto the chromosomes of S. cerevisiae . This new homolo-gous recombination method allows sequential cloned geneinsertions into dispersed target si tes in the genome.

A model inducible expression cassette, CUP1p-lacZ ,w a s u s e d t o e v a l u a t e t h i s s y s t e m . Af t er f i v e r ou n d s o f

transf ormation, U R A 3 curing, and S outhern b lot ana ly-s is , a s er ie s o f s t r a i n s w i t h on e t o f i ve n on t a n d e mintegrations wa s ob ta ined. Gene expression wa s foundto be extremely w ell (linearly) correla ted w ith int egrat edcopy num ber. The level of expression for one integra tedgene wa s a lso compar ab le to tha t f or one copy of a CE N/ARS pla smid carr ying th e same expression cassett e (Leea n d D a S i l va , 1 99 6b ). Th e se r e s u lt s d e m on s t r a t e t h epotential of this method for the introduction of multipledif ferent genes (e.g., for meta b olic engineering), withsub sequent expression of each gene related to i ts copynumb er. In a ddition, the integrations (at least up to fivecopies per cell) did not n ota bly a ffect cell growth . Thestructural sta b ili ty wa s found t o b e location-dependent;the sta b ili t ies of the f ive integra tions w ere 75, 85, 100,

100, an d 100%a fter 50 genera tions in complex medium.B l i n d s e q u en t i a l t r a n s f o r m a t i on s (w i t h o u t S o u t h er nan a lysis aft er each insertion) were only successful for th einsertion of small numbers of genes.

The S. cerevisiae Ty1 a nd Ty3 retrotransposons cana l s o b e u s ed f or t h e i n se rt i on of cl on ed g en es i nt odispersed sites in the chromosomes (Boeke et al. , 1988;Cha lker a nd Sa ndmey er, 1992). This method exploits thereplicat ion (via reverse t ran scription) a nd integrat ionmecha nisms of the na tive Ty elements. Our lab oratoryha s a lso focused on developing an d evalua ting sequent ialintegration vectors using Ty1 and Ty3 elements (Lee andD a S i lv a , 1996a , b ; Wa n g a n d D a S i lv a , 1996). R et -rotransposition and homologous recombination are fun-d a m e n t a l l y d i ff er e n t a n d h a v e d i s t i n ct a d v a n t a g e s a n d

disadva nta ges. Retrotra nsposon-mediat ed integra tion isvery promising a nd is a t ruly regula ted meth od of clonedgene integra tion. However, it is a lso more complex andrequires a reverse t ra nscription step; size l imits (f orsuccessful tra nsposition) on the inserted cloned genecassettes ma y a lso exist . Like Ty-mediated integra tion,the new δ/U B method can be used to sequentia lly insertg e n es . H o w e ve r , i t i s a l s o m u ch m or e e a s i l y i m p le -mented, a nd a size l imita tion on the inserted gene(s) isnot a concern. Interestingly, b oth m ethods resulted instra ins w ith a pproximat ely the sa me expression level per

integrated CUP1p-lacZ gene; -gala ctosidase activi t iesfor stra ins car rying one t o five Ty1-integr a ted copies (Leea n d D a S i l va , 1 99 6b ) w e re s i m i la r t o t h o s e s h ow n i nFigure 5. In b oth cases, f ive integrated copies resultedin a pproximat ely 50% of the a ctivity measured f or thesame cassette on a 2 µ-b ased plasmid (Lee and D a Silva,1996b).

While our r esults demonstra te the ef fectiveness ofδ/UB -integrat ion for repeti t ive integrat ions of clonedg e n es , f u r t h er i m pr ov em e n t i s p os s ib le i n t w o a r e a s .Increasing int egrat ed copy number w ould be significan tlyeasier i f tandem insertions (next to integrations f romprior t ran sforma tions) could b e avoided. In addition, i tw o u ld b e p r e fe r a b le i f a l l i n s er t i on s w e r e ca . 1 00 %structura lly sta ble during long-term cultivation. A modi-

f ic a t i on w h i ch s h ou l d a l l ow t h e s e i m pr ov em e n t s (a l -though at the expense of reduced integration frequency)is to insert the expression and U R A 3 B l a s t e r c a s s e t t esw i t h in t h e δ sequence a nd rely on a double-crossoverevent for integrat ion. The construction a nd a na lysis ofth is double-crossover vector and furt her modificat ions fora p p li ca t i on i n p ol y pl oi d y e a s t s t r a i n s a r e cu r r en t l yunderway in our lab oratory.

Acknowledgment

Th e a u t h or s a r e g r a t e f u l t o S u z a n n e B . S a n d m e ye r(University of Ca li fornia, Irvine) for providing str ainTM y16, J ef D. Boeke (J ohn Hopkins Un iversity) f orp la s m i d p H 3, a n d M ich a e l G r u n s t e i n (U n i v er s it y of

Ca lifornia , Los Angeles) for plasm id pLD3. This work w a ssupported in part b y the National Science Foundation(G ra nt Nos. B ES -9119808 and B CS -9311021).

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samples with N copiesremaining

originalintegrated

copy no.g r o w t h

mediumsample

size N ) 5a N ) 4b N ) 3b

5 YP D 30 2 3 5 25 YP D + 1 m M

C u S O4

30 20 7 3

a Based on ONPG tests (two-thirds of the samples were verifiedvia Southern analysis) . b B a s e d o n S o u t h er n a n a l y s i s.

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Accepted J une 4, 1997.X

B P 970055D

X Abstract published in A dvance ACS Abstracts, J uly 1, 1997.


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