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Improved E rythromycin Production in a Genetically EngineeredIndustrial Strain ofSa cch a r op ol yspor a er yt h r a ea

Wolfgang Minas, Peter Bru1nker, Pauli T . Kallio, and J ames E. Bailey*

Ins tit ute of B iotechnology, E TH-Zu rich, C H-8093 Zurich, S w itzer lan d

An indust rial eryt hromycin production str ain of Sacchar opol yspor a eryt hr aeaspp. wa su s ed to d emon s tr a t e th a t ca r ef u l gen etic en gi n eer in g ca n s i gn i fi ca n t l y i mpr ov eproductivity . The chromosomally integra ted V itr eoscil la hemoglobin gen e (vh b) w a sshown t o enha nce the final t iter of erythromycin by some 70%compar ed to the originalS. erythraea spp. Overa ll , specific eryth romycin yields were about 2.5 g of eryth ro-mycin/g of t ota l pr otein for S. erythraea::vhb bu t

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U n l e s s o t h er w i s e s t a t e d , a l l c h em i ca l s w e r e a n a l y t i ca lq u a l it y a n d ob t a i ne d f rom S i gm a , F l uk a , or M er ck .Medium M1 contains, per liter of reverse osmosis (RO)wa ter, 5 g of a nhyd rous glucose, 5 g of tryptone (Oxoid),0 . 5 g o f b e t a i n e - H C l , 5 g o f A R G O c o r n s t a r c h ( C P CInt erna tiona l Inc., Englew ood Cliffs, NJ ), 1 g of corn st eepliquor, 200 mg of MgSO47H 2O, 2 mg of ZnSO47H 2O, 0.8mg of CuSO45H 2O, 0.2 mg of CoCl26H 2O, 4 mg of FeSO47H 2O , 8 0 m g o f C a C l 26H 2O , 1 0 g o f N a C l , 1 5 0 m g o fK H 2P O4, a n d 2 0 g o f B a c t o Ag a r (D i f co). M ed i u m V 1contains per liter of RO water 16 g of ARGO corn starch(CP C), 10 g of dextrin (D-2256, S igma ), 15 g of soybeanflour (32H0411, S igma ), 2.5 g of Na Cl (Merck), 5 m L ofcorn steep liquor, 1 g of (NH 4)2S O4 (Rot h), 6 m L/L pu resoybea n oil (#01190 Nef Lebensm itt el AG, Zu rich, C H),a n d 4 g o f C a C O3 (Sigma ). The pH wa s a djusted to 6.5before aut oclaving. One liter of V2 medium, used for th esecond-stage inoculum, was made of 18 g of corn starch,12 g of dextrin, 15 g of soybean flour, 3 g of NaCl, 6 mLof corn steep liquor, 1.2 g of (NH 4)2S O4, 6 mL of soybea noil , and 5 g of CaC O3 suspended in RO wa ter . After themedium wa s a utoclaved the pH w as adjusted to pH 6.8.The eryt hromycin production m edium, F1, conta ined 35g of corn s ta rch, 32 g of dextrin, 33 g of soybean flour, 7g of NaCl, 20 mL of corn steep liquor, 2 g of (NH 4)2S O4,

6 m L of s o y be a n oi l, a n d 8 g o f C a C O 3 per l i ter of ROw a t e r . S t e r il e a n t i foa m , M a z u D F 204 (P P G O uv r ie ,Lesquin, Fra nce), w as added prior t o steri lization (0.5mL/L) and dur ing cultivat ions as needed. The pH of thesteri le F1 medium w as a djusted to pH 6.5. Since mediacontaining soyb ean f lour star ted foaming at around 70 C, causing t he exhaust air f i lter t o b ecome soiled whensterilized in bioreactors, V2 medium wa s st erilized in theb ioreactor without soyb ean f lour. The latt er wa s aut o-c l a v e d s e p a r a t e l y a n d a d d e d a s e p t i c a l l y t o t h e s t e r i l eb ior e a ct o r . Th e o r ig i n a l F 1 m e d iu m w a s u s ed a t h a l f -concentra tion only. All salts a nd dextrin were aut oclavedseparately and were added aseptical ly to the steri l izedbioreactor which contained all of the other components.Due t o the h igh portion of undissolved ma terial, st eriliza-

tion t imes for b ioreactors were increased to 50 min at121 C.Test medium, consisting of tryptic soy b roth (TSB ,

Oxoid) supplemented wit h 2 g/L glucose a nd 2% a ga r,was used for M . l u t eu s -based erythromycin bioassy.

Cultivations were performed in three stages accordingt o a p r ot o col p r ov i de d b y R . S w a r t z (p er s on a l com -municat ion). The f irst-sta ge seed culture wa s grown in35 m L of V 1. Af t er 48 h , t h i s c ul t ur e w a s u s ed t oinocula te 3.5 L of V2 medium. This second-sta ge seedcultivation was performed in a 5-L LH 210 b ioreactor(Inceltech LH SG I S .A., Fra nce) equipped w ith a pitchedb lade turb ine. The agita tion speed w as set to 800 rpm;t h e a i r fl ow r a t e t o 0 .6 v vm ; t h e t e m per a t u re w a scontrolled at 34 C; and the dissolved oxygen tension

(DOT), pH, a nd redox potent ial profiles w ere monitored.C O2 a n d O2 co nc en t r a t i on s i n t h e e x h a u s t g a s w e r emonitored on-line with a mass spectrometer (VG Prima600, VG G as Analysis S ystems, Middlewich, U.K.). After40 h, 1.5 L of culture b roth was transferred to a 20-LInfors ISF200 b ioreactor (Infors AG, Bottmingen, CH)equipped with two Rushton turb ines and containing 10L of half-strength F1 production medium. Cultivat ionconditions w ere set a s fol lows: the temperature w as setto 34 C; t he pH controlled w ith H 2S O4so as n ot to exceed7.2; the agita tion speed wa s init ial ly set t o 700 rpm a ndcontrolled b y t he DOT signal t o increase t o a ma ximumof 900 rpm if th e DOT fell below 45%air sa tura tion; theair f low rate was set to 0.37 vvm for the f irst 12 h andthen cha nged to 0.83 vvm, which fel l to 0.7 vvm as thefeed w as a dded to the reactor volume; and the pressurew a s s et t o 0. 1 ba r . F e ed in g r a t e s a n d d u r a t i on s w e r e2.4 mL /(L/da y) n-propa nol from 12 to 160 h, 4.8 mL/(L/da y) soybean oil from 25 h unt il the end of the cultivat ion,a nd 48 mL/(L/da y) 15%dextrin from 30 t o 90 h. The lowf low r a t e s w e r e r e a l i ze d b y a c t i v a t i n g t h e f ee d p u m p son ce p er m i n ut e t o a d d t h e p r op er a m o u n t s of f ee dcompounds. Redox potent ial an d CO2a n d O2concentra -t i on s i n t h e e xh a u s t g a s w e r e m o n it or ed on -l in e a sdescribed above. In a ddition, free glucose wa s measured

hourly w ith a YSI 2700 Biochemistry Analyzer fitted wit hthe 2730 Monitor and Control Module (YSI Inc., YellowSprings, OH). Sa mples were dra wn a septical ly througha cross-flow filter assembly (BioEngineering, Wald, CH)f it t e d w i t h a 0. 2-m I R I S 6502 m em b ra n e (P h o n e-P oulenc Tech-Sep, Miribel, Fr a nce). Da ily sam ples of 50or 100 mL were drawn to determine the erythromycintiter and for CO difference spectroscopy, Western Blotanalysis, and microscopic inspection f or b oth possib leconta minat ion an d mycelial morphology. Sa mples werestored at -2 0 C .

A s t a n d a r d b i o a s s a y w a s a p p l i e d t o d e t e r m i n e t h eerythromycin t i ters using erythromycin (Fluka) as sta n-d a r d (17).

Since the media contain large amounts of unsolub le

m a t e r i a l w h i ch w o u ld i n t er f er e w i t h a d i r ec t c e ll d r yweight determination, b ioma ss changes were monitoredb y measuring the total protein content of the samplesby a standard Folin reagent procedure (18) af ter alka linelysis of culture samples (19). B o v in e s e r um a l bu m i n ew a s u s e d a s a s t a n d a r d .

Th e H P L C a s s a y f o r t h e d e t e r m in a t i o n o f t h e e r y t h -romycin composition from culture broth was describedearl ier (20).

The C O difference spectroscopy a ssa y a nd Western blotan alysis ha ve b een d escrib ed elsewhere (21, 22). H o w -ever, prepara tion of the sam ples required some at tention.Following cell disruption using a French press (Aminco,SLM Instruments Inc. , Urb ana, IL), an ultracentri f uga-tion st ep of 4 h at 150000gin a SW41Ti rotor (Beckma nn)

Table 1. Reports on the Effects ofV i tr eosci l l a Hemoglobin Gene Functionally Expressed in Different BiologicalSystems

biologica l sy st em effect s r ef

Escherichia coli enhanced growth and recombinant protein productionunder oxygen-limited conditions

5, 6, 7

St r eptom yces coeli col or in cr ea sed a ct in or h odin biosy n t h esis 8Acremonium chrysogenum in cr ea sed ceph a lospor in C pr oduct ion 9Corynebacterium glutamicum increased L-ly sin e pr oduct ion 10B a c i l l u s s u b t i l i s increased total protein secret ion, neutral protease act ivity,

a n d R-amy lase a ct ivity11

X a n t h o mon a s ma l t o p h i l i a bior em edia t ion of ben zoic a cid 12C hin ese h am st er ov ar y cells en ha n ced pr od uct ion of h um a n t issu e pla sm in ogen a ct iv at or 13Sacchar omyces cer evi siae in cr ea sed a er obic sy n t h esis of et h a n ol 14t oba cco in cr ea sed n icot in e con t en t 15

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was used to separate soybean oil, soybean oil-bound smallparticles, and cell debris from the cleared cell extract.

Results

Bioreactor fed-batch cultivations were performedusing both the original S. erythr aeaa nd th e metabolicallyengineered strain, S. erythraea::vhb, w h i c h c o n t a i n s asingle copy of the vh b gene integrat ed into i ts chromo-some via the a t tsi te f rom pha ge C31 (16). Because thevh b gene a nd i ts expression were st ab le in shake-f laskcultivat ions (16), no thiostrepton was added to the fed-bat ch fermenta tions. These experiments w ere conductedat the 10-15-L s cale; a useful representa tion of th e pilotscale w hich a lso permits larger sa mples to b e withdra wn

required monitoring vh b expression b y Western b lotan alysis a nd VHb a ctivity by CO difference spectroscopy.As described in the experimental protocol, the second-sta ge preculture wa s performed in a b ioreactor . Thisimproved t he reproducibility of th e V2 cultivat ions. ThepH and redox potential profiles for duplicate and tripli-cate cultivations were almost identical for each of thestra ins. During the f irst 16 h, the pH dropped to almost6 and rema ined low unt il after 16 h, when it rose sharplyto reach its ma ximum of 8 at around 24-26 h, after whicht h e p H f e ll a g a i n t o v a l u es a b ou t 6 . 5 a t 4 0 h . D u r i n gp r od u ct i on i n h a l f-s t r e n g t h F 1 m e d i u m , t h e p H w a scont rolled not t o exceed 7.2. Ferment a tion profiles fromS. erythraea s p p . a n d S. erythraea::vhb a r e s h o w n i nFigure 1. The horizonta l b ars in the center indicat e the

dura tion of the respective feeds. It is interesting to notetha t, w ith t hree exceptions, almost no free glucose couldb e d e t ect e d . Th e f i r st a n d g r ea t e s t i n s t a n c e o f f re eglucose, a bout 1 g/L, occurred during th e first 12 h a fterinoculat ion, t he second af ter 38 h following the st a rt ofthe dextrin feed, and the third at the end of the dextrinfeed.

Differences between the recombinant S. erythr aea::vhba n d t h e o r i g i n a l S. erythraea spp. were also ob served.These include the higher concentration of free glucose,0.4 g/L, a fter 38 h in the S. erythraea spp. cultivation,compared to 0.25 g/L w ith t he recombinan t st ra in a s w ellas the a ppeara nce of the th ird peak only for the originalstra in. This tra nsient peak of free glucose coincided wit ha decrease in the erythromycin production rate. Int er-estingly, biomass da ta , expressed as tota l protein per liters h o w e d t h a t S. eryth raea::vhb ma kes ab out 24% lessbiomass compared to the control (Table 2), indicating aslower growt h ofS. eryt hr aea::vhbw ith greatly increaseds p eci fi c m e t a b ol ic a ct i v it y . F i g ur e 2 s u m m a r i z es t h eresults of f ive such b ioreactor cultivat ions: three f er-mentations of S. eryth raea spp. and two cultivations ofthe recomb inant S. erythraea::vhb. Th e f in a l e r y t h r o -mycin t iters obta ined a vera ged 7.25 g/L for t he recom-b inant S. erythraea::vhb a nd 4.25 g/L for the originalproduction st ra in. This corresponds t o an increase inerythromycin volumetric yields of a b out 70%. Thisi ncr ea s e i n p rod u ct i vi t y w a s t h e r es u lt of a h i gh ererythromycin b iosynthesis ra te. The production ra tes

were calculated for the t ime interval f rom 48 to 144 ha nd found t o be 57.5 mg/(L/h) for th e recombina nt , vh b-expressing strain, S. erythraea::vhb, compared to only24.3 mg/(L/h) for t he original production str a in. Thehigher erythromycin production t ogether with 24-40%lower biomass concentra tion obta ined with S. erythr aea::vh b resulted in an about 2.5-fold higher overall specificeryt hromycin y ield (g of eryt hromycin/g of protein) in th isrecombinant stra in (Tab le 2). HP LC a na lysis of b rothsamples revealed t ha t the ra tios of erythromycin A (theactive compound) to erythromycin B an d C were identicalfor the t wo stra ins (dat a not shown). M icroscopic ana ly-sis of the samples revealed that , star ting at ab out day 8(192 h), a progressive fra gmenta tion of the myceliumocc ur r e d. D i s in t e g r a t i on o f t h e m y ce li u m w a s m or e

pronounced in S. erythraea spp. than in the recombinantS. eryth tr aea::vhb.

Syn thesis a nd a ctivity of the het erologous VHb proteinin S. erythraea::vhbw e r e a n a l y ze d t o d e m on s t r a t e t h a tthis cha nge in production is correlated w ith t he expres-sion of vh b i n t h e r e com b in a n t s t r a i n . We s t er n b lo t sw e r e m a d e w i t h s a m p le s f r om cu lt i va t i on s w i t h t h er e com b in a n t a n d t h e o r ig i n a l s t r a i n . A cl ea r b a n d t h a tcorresponds to the posit ive control was detected in al ls a m p le s f rom t h e S. eryth raea::vhb t h r ou g h ou t t h ecultivat ion while no VHb -specif ic b an d wa s detected insamples from cultivations with S. erythraea spp. (datanot shown).

The activi ty of VHb was examined b y CO dif f erencespectroscopy. CO difference spectra from S. erythraea

Figure 1. D a t a f r om 10-15L bioreactor fed-batch cultivationsi n h al f -st r e n g t h F 1 p r od u ct i on m e d iu m of S. erythraea spp.(upper panel) and S. erythaea::vhb(lower panel). Total proteinm e a s u r e m e n t s o f t h e s a m p l e s a r e s h o w n a s b a r s w i t h t h e i rrespective values in gra ms per l i ter. The horizonta l bars in themiddle represent the duration of the indicated feed phases.

Table 2. Summary of Data on Biomass (Total Protein)and E rythromycin Yield of the Two Cultivations Shownin Figure 1

s t r a i nt i m e

(h)

t o t a lprotein

(g/L )

erythromycintiter(g/L )

erythromycinyield on

biom a ss (g/g)

S. erythraea::vhb 168 2.6 6.7 2.6216 3.2 7.4 2.3

S. erythr aeaspp. 168 4.1 3.6 0.9216 4.2 4.0 1.0

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spp. show a peak a t 450 nm. This indica ted th e presenceof cytochome(s) P-450 in this strain and probably is dueto the cytochrome P-450 monooxygenases E ryF an d E ryKof the erythromycin gene cluster (23). The spectra fromS. erythraea::vhb s h ow a n a d d i t ion a l p ea k a t 4 20 n mchara cteristic of active VHb wh ich is detected t hroughoutthe cultivat ion (Figure 3). M oreover, the cytochromeP-450 peak is ab out 5 t imes lar ger in S. erythraea::vhbt h a n i n S. eryth raeaspp. , indica ting an increased levelof P -450 cyt ochromes.

Discussion

G enetic engineering, applied to modif y meta b olismad va nta geously, ha s been proposed a s the ma in route for

further significan t improvements on industr ia l produc-tion strains (24). Thus far , recombina nt D NA techniquesw e r e u t i l iz ed on on l y a f ew s t r a i n s o f e r y t h r om y ci nproducingS. erythr aeain a ttempts to improve productiv-i t y. M os t of t h es e s t r a i n s a r e a v a i l a b le f r om s t r a i ncollections an d are not representat ive, genetical ly orphysiologically, of the highly mutagenized and selectedstra ins used f or industr ia l a ntib iotic production (1, 25).Furt hermore, these genetic modifications often increasedt h e s t r a i n s g e n et i c i n s t a b i li t y s u ch t h a t co nt i n u ou sselection pressure w as required to ma inta in t he recom-b in a n t s t r a i n s d u r in g t h e c u lt i v a t i on s (25). Assumingt h a t t h e S . er y t hr aea s pp . s t r a i n h a d a l r ea d y b ee nselected for a highly productive configuration of eryth-romycin b iosynthesis genes (er y), a ny modif icat ion of

these genes could increase genetic instab il ity or resultin a loss of productivity (26). Int roducing a heterologousg en e, h ow e ve r, t h a t h a d p re vi ou s ly b ee n s h ow n t oimprove cell growth and protein production in a varietyof biological systems (Table 1) seemed a more promisinga pproa ch to increa se the productivity of our S. erythraeas t r a i n .

While the biosynthesis ra te in S. erythraeaspp. d roppedaft er about 90 h into the cultiva tion from initial high ra teto a reduced rat e of 1.9 mg/(L/h), the erythromycinproduction rate with S. eryth raea::vhb r e m a i n e d a t t h ehigh ra te of 4.2 mg/(L/h) (Figure 2). The combina tion off a s t e r e r y t h r om y ci n p r od u ct i on w i t h co nt i n u a t i on ofproduction to higher t i ters b y S. eryth raea::vhb affordseven greater advantages in overall space-time yield of

t h e p r oce s s. F o r e xa m p l e, b y h a r v e s t in g t h e S. eryth- raea::vhb a f t e r 1 44 h , a s pa c e-t i m e y i e l d o f 1 . 1 g o feryt hromy cin/(L/da y) is achieved , wh ich is 100%grea tert h a n t h e s p a ce-time yield a chieved by S. erythraea spp.ferment a tion (0.56 g of eryt hromy cin/(L/da y), ha rvest ingat the point of maximum ti ter a f ter 192 h).

It was interesting to ob serve that the recomb inant S.

erythraea::vhb made signif icantly less b iomass while att h e s a m e t i m e-c on s u m in g g l uc os e m or e r a p i d ly a n dproducing 70%more erythromycin. This indicates t hatspecif ic metab olic activi t ies in this strain were greatlyincreased relative to its predecessor.

H ow t h e p re se nce of VH b cou ld a f f ect g r ow t h ore r y t h r om y ci n p r od u ct i on i s n o t c e rt a i n . I t h a s b ee np r op os e d t h a t V H b i m pr ov es ox y g en a v a i l a b i li t y b yincreasing intracellular oxygen concentration under mi-croaerobic culture conditions (27, 28). D e s pi t e a h i g h(>45% of a ir sat urat ion) dissolved oxygen (D O) levelthroughout the cultivations, oxygen might be limited inthe mycelial aggregates in the rather viscous productionmedium.

In a ddition, it ha s been shown forEscheri chia coli t h a t

under oxygen limitation expression of the cytochrome otermina l oxidase is repressed through th eF N Ra n d A R Csystems (29 ). Expression of VHb upregulat es cyt ochromeo terminal oxidase expression up to 5-f old in E . c ol i ,probably by increasing the intracellular oxygen concen-t r a t i on (27, 30). VHb expression in S. erythraea::vhbcould have a related ef fect , since i t is reasonab le thatexpression of P -450 cytochromes could increa se a t h igheroxygen levels. Ava ilability of these cytochromes couldinfluence eryth romycin production. How ever, previousstudies have indicated that specific erythromycin produc-tion ra tes rema in unchanged under a erob ic, 60%D OT,microana erob ic, 10% DOT, or even an aerob ic cultureconditions (31, 32). These results, however, w ere ob-ta ined with a much less developed stra in (100-200 mg

Figure 2. Ave r ag e d e r y t h r om y c in t i t e r s a s obt a i n ed i n 10-15-L bioreactor fed-bat ch cultivations. Mean s a nd error bars ofduplicate a nd tr iplicat e cultivat ions are shown for S. eryt hr aeaspp. (circles) an d S. er ythaea::vhb (squares), respectively.

Figure 3. CO dif ference spectra from cleared cell lysates insa mples from 10-15-L bioreactor fed-batch cultivations. The flatbaseline (dotted l ine) represents sa mples prior to CO binding.Af t e r e xp osu r e t o C O, t h e sam p l e s f r om S. ery th r a ea spp.cultivations (day 4 sa mple shown, da shed line) show a clear peaknear 450 nm which is probably due to the P-450 monooxyge-nases encoded by eryF1 a n d eryK o f t h e e r y t h r om y c i n g e n ecluster. CO-treated samples from cultivations with S. eryt hr aea::vh b (day 8 shown, solid l ine) show, in addition to the 450-nmp eak , t h e V Hb -sp ec if ic p e ak at 420 n m d e m on st r at i n g t h epresence of active VHb in the cells. The plot scale is compensatedfor dif ferences in t he sa mples protein contents.

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of erythromycin/L) on def ined medium tha t might notadequa tely represent t he production st rain, S. erythraeaspp. , and process used in this st udy.

Eryt hromycin b iosynthesis involves the action of tw ocytochrome P-450 monooxygenases, encoded by eryF1a nderyK. EryF performs th e C6-hydroxylat ion w hich con-verts 6-deoxyerythronolide B to erythronolid B (23, 33),while EryK is required for the f inal hydroxylation stepconverting erythromycin D to erythromycin C or eryth-romycin B to erythromycin A (33, 34). This opens the

possib il i ty of direct interaction b etween VHb and themonooxygenases during eryt hromycin biosynt hesis. Re-ferring aga in t o previous research on VHb ef f ects in E .coli, the specific activity of cytochrome o was increased50%in cells expressing VHb (30).

Acknowledgment

We thank Professor Randall Swartz for assistance withthe fermentation process. This work was supported b ySolidago AG and Swiss KTI Project No. 3034.1.

References and Notes

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(2) Roman, R. V.; Ga vrilescu, M. Oxygen t ra nsfer eff iciency inthe biosynthesis of ant ibiotics in bioreactors w ith a modif iedR u sh t on t u r b i n e ag i t at or . Acta Biotechnol. 1994, 14, 181-192.

(3) Webster, D. A. Structure and function of bacterial hemo-globin and related proteins. Adv. Or g. Biochem. 1988, 7, 245-265.

( 4) Wak ab ay ash i , S . ; M at su b ar a, H. ; We b st e r , D . A . P r i m ar ysequence of a dimeric bacterial ha emoglobin fromVi tr eoscil la.N a t u r e 1986, 32 2, 481-483.

(5) Kh osl a , C . ; B a i le y , J . E . He t e r ol og ou s e xpr e ssion of a

b ac t e r ial h ae m og lobi n i m pr oves t h e g r owt h p r op er t i e s ofrecombinant Escherichia coli. N a t u r e 1988, 33 1, 633-635.

(6) Kh osla , C . ; C u r t i s , J . E . ; D e M ode n a, J . ; R i n as, U . ; B ai l e y ,J . E. E xpression of intr acellular hemoglobin improves proteinsynthesis in oxygen-limited Escherichia coli. Bio/ Technology1990, 8, 849-853.

( 7) Kh osr avi , M . ; We b st e r , D . A . ; S t ar k , B . C . P r e se n c e ofbacterial hemoglobin gene improves R-amylase production ofa r e com bi n an t Escheri chia coli s t r a i n . Pla s mid 1990, 24,190-194.

(8) M ag n ol o, S . K. ; L e en u t ap h on g , D . L . ; D e M od e n a, J . A. ;C u r t i s, J . E . ; B a i l ey , J . E . ; G a l a z z o, J . L . ; H u g h e s, D . E .Actinorhodin production by Streptomyces coelicolorand growthof Streptomyces lividans are improved by the expression of abacterial hemoglobin. B io/ T echnology 1991, 9, 473-476.

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Accepted J une 8, 1998.

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