Temperature- and salinity-decoupled overproduction of hydroxyectoine by chromohalobacter salexigens

Tempe a u e- and Salini y-Decoupled O e p oduc ion o Hyd oxyec oine by Ch omohalobac e salexigens Ja ie Rod íguez-Moya, a Mon se a A gandoña, a Fe nando Iglesias-Gue a, b Joaquín J. Nie o, a Ca men Va gas a Depa men o Mic obiology and Pa asi ology a and Depa men o O ganic and Pha maceu ical Chemis y, b Facul y o Pha macy, Uni e si y o Se ille, Se ille, Spain Hyd oxyec oine o e p oduc ion by he na u al p oduce Ch omohalobac e salexigens is p esen ed in his s udy. Gene ically enginee ed s ains we e cons uc ed ha a low salini y coexp essed, in a ec o de i ed om a na i e plasmid, he ec oine (ec ABC) and hyd oxyec oine (ec D) genes unde he con ol o he ec A p omo e , in a empe a u e-independen manne . Hy- d oxyec oine p oduc ion was u he imp o ed by inc easing he copies o ec D and using a C. salexigens gene ic backg ound unable o syn hesize ec oines. Ec oine and hyd oxyec oine (ec oines) a e compa ible solu es syn hesized and accumula ed by halophilic and halo ole an bac e ia in esponse o osmo ic and hea s ess (1,2). Ec oines ha e cu en applica ions as bios abilize s o p o eins and nucleic acids, as well as a po en ial ole as he apeu ics o ce ain diseases (3,4). This, oge he wi h he complexi y o hei chemical syn- hesis, has encou aged ecen e o s o imp o e ec oine p oduc- ion om bac e ia. Hyd oxyec oine is especially in e es ing, as i seems o con e addi ional p o ec ions de i ed om i s hyd oxy- la ed na u e (3,4). Ec oines a e syn hesized om aspa a e semi- aldehyde. Fi s , his me aboli e is con e ed in o diaminobu y ic acid, which is ace yla ed o N␥-ace yldiaminobu y ic acid and subsequen ly cycled o ec oine (5,6). The main ou e o hy- d oxyec oine syn hesis is ia ec oine hyd oxyla ion (2). The en- zymes o ec oine syn hesis a e usually encoded in an ec ABC- ype gene clus e , which is usually well conse ed among ec oine-p o- ducing mic oo ganisms (7). The e a e excep ions, such as incom- ple e ope ons, gene clus e s including he ask gene ( o he aspa - a e kinase), gene clus e s ca ying ec ABC-ec D-ask, and sca e ing o he genes wi hin he ch omosome, wi h duplica ions o ec C and ec D o e en soli a y ec C (5,7,8). Indus ial p oduc- ion o hyd oxyec oine uses Halomonas elonga a ATCC 33173 T g own unde high-salini y and high- empe a u e condi ions, us- ing he bac e ial milking me hod (9), o a de i a i e o his ech- nique (10), ollowed by sepa a ion and pu i ica ion o ec oine and hyd oxyec oine (9). This sal and empe a u e equi emen o hyd oxyec oine syn hesis is a se ious d awback o using na u al p oduce s, since e men a ion unde high empe a u e and salin- i y inc eases p oduc ion cos s and co odes indus ial eac o s. Ch omohalobac e salexigens is a halophilic gammap o eobac- e ium which p oduces ec oine and hyd oxyec oine in esponse o sal and hea s ess, espec i ely (7). I is easy o g ow and i s genome sequence is a ailable (h p://genome.o nl.go /mic obial /csal/). I has been sugges ed as an al e na i e o H. elonga a (11). In C. salexigens, he genes encoding ec oine syn hesis lay wi hin a 2.8-kb egion encoding he diaminobu y ic acid ace yl ans e ase (Ec A), diaminobu y ic acid ansaminase (Ec B), and ec oine syn hase (Ec C) (12). The mic oo ganism has wo pa alogs o he enzyme ec oine hyd oxylase, Ec D and Ec E, bu Ec D is he main esponsible enzyme o hyd oxyec oine p oduc ion (2). In C. salexigens, he gene clus e ec ABC and he genes ec D and ec E a e a di e en loci wi hin he ch omosome. Whe eas accumula ion o bo h solu es in C. salexigens is max- imal du ing s a iona y phase, he accumula ion o hyd oxyec oine is up egula ed by salini y and empe a u e, and he accumula ion o ec oine is up egula ed by salini y and down egula ed by em- pe a u e. Thus, hyd oxyec oine p oduc ion and accumula ion is maximum a 45°C and 14.5% NaCl, while ec oine accumula ion eaches i s maximum a 37°C and 17.4% NaCl (2). This egula ion occu s, a leas in pa , a he ansc ip ional le el. The ec oine syn hesis genes ec ABC can be exp essed om wo p omo e e- gions, one loca ed ups eam o ec A and composed o ou pu a- i e p omo e s (Pec A1 o Pec A4 [Pec A1-4]) and a second in e - nal p omo e loca ed ups eam o ec B (Pec B). In silico analysis o he ⫺10 and ⫺35 sequences o hese egions showed ha Pec A1 and Pec A2 may be dependen on he main ege a i e ac o ␴ 70 (and he e o e cons i u i ely exp essed), whe eas Pec A3 and Pec B we e simila o ␴ S - and ␴ 32 -dependen p omo e s, espec- i ely. In ag eemen wi h hese p edic ions, exp ession o a Pec A1-4::lacZ usion was osmo egula ed and depended in pa on he gene al s ess ac o ␴ S , whe eas Pec B was induced by con in- uous g ow h a a high empe a u e (13). On he o he hand, he p omo e egion o ec D is composed o wo p omo e s (Pec D1 and Pec D2), and ec D exp ession is bo h osmo- and he mo egu- la ed (M. Reina-Bueno, unpublished da a). In his wo k, we ha e me abolically enginee ed C. salexigens o o e p oduce hyd oxyec oine a low salini y, in a empe a u e-in- dependen manne . In o de o maximize hyd oxyec oine p o- duc ion and o minimize i s empe a u e and salini y equi e- men s, we designed ansc ip ional usions be ween he ec oine syn hesis genes ec ABC and he main hyd oxyec oine syn hesis gene ec D, so ha he second became ansc ip ionally con olled by he ec ABC p omo e egion. To cons uc a unc ional ec ABCD casse e, we i s ampli ied by PCR a 3,384-bp sequence, including he p omo e egion ups eam o ec A, he ec ABC gene clus e , and he ho-independen e mina o downs eam o ec C, Recei ed 8 Sep embe 2012 Accep ed 9 No embe 2012 Published ahead o p in 16 No embe 2012 Add ess co espondence o Ca men Va gas, [email p o ec ed]. Supplemen al ma e ial o his a icle may be ound a h p://dx.doi.o g/10.1128 /AEM.02774-12. Copy igh © 2013, Ame ican Socie y o Mic obiology. All Righ s Rese ed. doi:10.1128/AEM.02774-12 1018 aem.asm.o g Applied and En i onmen al Mic obiology p. 1018–1023 Feb ua y 2013 Volume 79 Numbe 3 on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om and cloned i in o pBluesc ip SK, esul ing in pME2. Then, we inse ed a BamHI es ic ion si e be ween ec C and he ho-inde- penden e mina o by si e-di ec ed mu agenesis using he p ime pai ec Bam_ w and ec Bam_ (see Table S1 in he supplemen al ma e ial o a lis o p ime s used in his s udy). Subsequen ly, we elimina ed he BamHI es ic ion si e om he mul icloning si e o pBluesc ip SK using he p ime s Qui Bam_ w and Qui Bam_ , ge ing pME2.3. Nex , we ampli ied a 1,200-bp sequence om he C. salexigens genome, including ec D, and inse ed i in o pBluesc ip SK, ge ing he plasmid pECTD. Then, we in oduced a BclI es ic ion si e downs eam o ec D using he p ime s ec DBcl_ w and ec DBcl_ , esul ing in pECTD2. Subsequen ly, we excised p omo e less ec D om pECTD2 by diges ing i wi h BclI and inse ed i in BamHI- diges ed pME2.3, yielding pECTABCD. Finally, he enginee ed ec ABCD gene clus e was excised om pECTABCD by diges- ion wi h EcoRI and cloned in o EcoRI-diges ed pHS15 (a cloning and exp ession ec o based on a na i e plasmid om H. elonga a ha bo ing a s ep omycin esis ance gene [14]), ob aining pHYDROX1 (Fig. 1A). To inc ease he ec D gene dose, a second unc ional casse e, ec ABCDD, was cons uc ed. Fo his pu pose, a second copy o ec D was cloned be ween ec D and he ho-independen e mina o in he p e ious ec - ABCD-cons uc ed gene clus e . Fi s , we in oduced a BamHI es ic ion si e be ween ec D and he ho-independen e mi- na o in pECTABCD, using he p ime s ABCDBam_ w and ABCDBam_ , ge ing pME2.4. Then, an in e nal BamHI si e o ec D was elimina ed by in oducing a silen mu a ion wi h he p ime s Qui BamD_ w and Qui BamD_ , esul ing in pME2.5. Nex , ec D was excised om pECTD2 wi h BclI and cloned in BamHI-diges ed pME2.5, esul ing in pECTABCDD. Subsequen ly, he ec ABCDD syn he ic gene clus e was excised by diges ion wi h EcoRI om pECTABCDD and cloned in EcoRI-diges ed pHS15, esul ing in pHYDROX2 (Fig. 1B). Bo h plasmids pHYDROX1 and pHYDROX2 we e ans- o med in o Esche ichia coli DH5␣cells, and he esul ing s ains we e used as dono s in a conjuga ion wi h C. salexigens wild ype and mu an CHR137 (⌬ec ABC::Tn1732,ec D::⍀; unable o syn hesize ec oines) (2). To e alua e hyd oxyec oine p oduc ion in bo h he na u al p oduce C. salexigens and he he e ologous hos E. coli DH5␣,we de e mined he ec oine con en in all s ains con aining pHY- DROX1 and pHYDROX2. Fo his pu pose, cells we e g own a di e en empe a u es (37°C o E. coli and 37, 40, and 45°C o C. salexigens) in shaking lasks wi h minimal medium M63 added wi h 20 mM glucose and di e en salini ies (1%, 2%, o 3% NaCl o E. coli s ains and 4.35%, 8.7%, o 14.5% NaCl o C. salexigens s ains) un il ea ly s a iona y phase. Cellula ex ac s used o liq- uid ch oma og aphy-mass spec ome y (LC-MS) and supe na- an s used o high-p essu e liquid ch oma og aphy wi h UV de- ec o (HPLC-UV) analysis o ec oine and hyd oxyec oine we e p epa ed by using a modi ied Bligh-Dye echnique desc ibed by K aegeloh and Kun e (15). Fo cellula ex ac s, ch oma og aphic sepa a ion and HPLC-elec osp ay ioniza ion was pe o med as desc ibed by A gandoña e al. (16). Fo supe na an s, samples we e analyzed as desc ibed by Ga cía-Es epa e al. (2). Hyd oxyec oine le els obse ed in he he e ologous ecombi- nan s ains E. coli DH5␣/pHYDROX1 and E. coli DH5␣/ pHYDROX2 g own a 1%, 2%, o 3% NaCl we e e y low, and only aces o ec oine we e de ec ed (da a no shown). Table 1 summa izes g ow h a es and ec oine and hyd oxyec oine p o- duc ion by he di e en C. salexigens wild- ype and ecombinan s ains, as well as hei speci ic p oduc ion a es. As p oduc ion o ec oines is di ec ly ela ed o he biomass p oduced a a ce ain salini y (11), he speci ic p oduc ion a e (␮mol/g bac e ial d y ma e [BDM] · h) is a simple unc ion e lec ing solu e con en and g ow h a e (17). As p e iously epo ed (2), ec oine accumu- la ion by C. salexigens wild ype was salini y dependen and, a a gi en salini y, in e sely co ela ed o inc easing empe a u e. Ec oine eached i s maximal accumula ion (725 ␮mol/g BDM) a 37°C wi h 14.5% o NaCl, wi h an ec oine/hyd oxyec oine a io o 2.24:1. On he o he hand, hyd oxyec oine accumula ion by he wild ype was salini y and empe a u e dependen , eaching i s maximum a 45°C wi h 14.5% NaCl, wi h an ec oine/hyd oxyec- oine a io o 0.45:1 and a yield o 942 ␮mol/g BDM. All C. salexigens ecombinan s ains ca ying plasmids pHYDROX1 o pHYDROX2, g own a 37°C o 40°C wi h 4.35% o 8.7% NaCl, showed much highe hyd oxyec oine and much lowe ec oine yields, espec i ely, han he wild- ype s ain ca y- ing no plasmid unde he same condi ions. In all cases, he majo p oduc was hyd oxyec oine, and he highe hyd oxyec oine p o- duc ion was accompanied by a dec ease o g ow h a es. This was co ela ed wi h he lowe ec oine yields obse ed, con i ming ha ec oine is necessa y o osmop o ec ion o C. salexigens. O he - wise, ec oine p oduc ion by C. salexigens ecombinan s ains a 14.5% NaCl could no be measu ed, as hey did no g ow o showed only esidual g ow h. Fo a gi en ecombinan s ain, g ow h a 40°C did no esul in a signi ican inc ease o hy- d oxyec oine yield, i compa ed o he same s ain g own a 37°C. This inding indica ed ha he highe hyd oxyec oine syn hesis in he enginee ed s ains was mos ly d i en by he Pec A p omo e egion and he e o e decoupled om empe a u e con ol. Wi h he excep ion o cells g own a 40°C wi h 8.7% NaCl, in he wild- ype backg ound he p esence o an ex a copy o ec D (i.e., cells ca ying pHYDROX2 e sus cells ca ying pHYDROX1 g own unde he same condi ions) did no imp o e hyd oxyec o- FIG 1 Gene ic o ganiza ion o cons uc ed inse s, cloned in pHS15 o ob ain pHYDROX1 (A) and pHYDROX2 (B). O e p oduc ion o Hyd oxyec oine by Ch omohalobac e Feb ua y 2013 Volume 79 Numbe 3 aem.asm.o g 1019 on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om ine yield. In addi ion, an inc ease in salini y (i.e., he same ecom- binan s ain g own a 8.7% e sus 4.35% NaCl a a gi en empe - a u e) did no imp o e hyd oxyec oine p oduc ion in he wild- ype backg ound. Howe e , in he ec oine-de icien s ain CHR137 (2), inc emen ing ec D dose o salini y did enhanced hyd oxyec oine yields. These indings sugges ha con ol mechanisms uled by en- dogenous ec ABC and/o ec D genes a e somehow ep essing hy- d oxyec oine syn hesis in he wild- ype backg ound. The bes p oduc ion esul s we e achie ed wi h s ain CHR137 ca ying pHYDROX2. This s ain g own a 8.7% NaCl eached a hyd oxyec oine yield o 967 ␮mol/g BDM (4.78- old highe han ha achie ed by he wild- ype s ain a his salini y), wi h a o al ec oine yield o 1,253 ␮mol/g BDM (ec oine/hyd oxyec oine a io o 0.29:1) and a signi ican dec ease o he g ow h a e (0.1 h ⫺1 ) ha educed he speci ic p oduc ion a e o 96.7 ␮mol/g BDM · h. Howe e , he same s ain g own a 4.35% NaCl showed a hy- d oxyec oine yield o 883 ␮mol/g BDM (14.96- old highe han ha obse ed in he wild- ype s ain a he same salini y), main- aining a easonable g ow h a e (0.14 h ⫺1 , wi h a speci ic p oduc- ion a e o 123.6 ␮mol/g BDM · h) and eaching an ec oine/ hyd oxyec oine a io o 0.1:1. These p oduc ion da a a low salini y and op imal empe a u e a e e y simila o hose shown by he wild ype g own a high salini y and high empe a u e (14.5% NaCl and 45°C). In mos bac e ia, he esponses o osmo ic and/o hea s ess in ol e he syn hesis o a cock ail o compa ible solu es (7). Thus, he p esence o by-p oduc s o he han ec oine was in es iga ed in he mos p omising s ain, CHR137/pHYDROX2, g own a 37°C wi h low salini y and compa ed o he cy oplasmic solu e pool syn hesized by he wild ype ca ying ei he pHYDROX2 o no plasmid. As shown in Fig. S1 in he supplemen al ma e ial, he majo compa ible solu e in C. salexigens wild- ype cells g own a 37°C wi h 4.35% NaCl was ec oine, ollowed by glu ama e and mino amoun s o glucosylglyce ol and hyd oxyec oine. In oduc- ion o pHYDROX2 in wild- ype cells swi ched he syn hesis o hy- d oxyec oine, which became he majo compa ible solu e, ollowed by glu ama e, ehalose, and mino amoun s o glucosylglyce ol and ec oine. The p esence o ehalose in he wild ype o e exp essing hyd oxyec oine a 37°C was unexpec ed, as his suga is syn hesized by C. salexigens in esponse o hea s ess o when ec oine is absen (18). This appa en induc ion o ehalose syn hesis by hyd oxyec o- ine will be in es iga ed in a u he wo k. Finally, ans e o pHY- DROX2 o he ec oine-de icien s ain CHR137 led o a much cleane compa ible solu e p o ile, consis ing o mainly hyd oxyec oine and educed amoun s o glu ama e and ehalose. Ec oine was no de- ec ed, al hough i should be p esen in mino amoun s, as judged by ou p e ious es ima ions (Table 1). In gene al, wo al e na i e biological sys ems would be sui able o app oach hyd oxyec oine p oduc ion: nonhalophilic mic oo - ganisms bea ing hyd oxyec oine syn hesis genes and na u al (halophilic) p oduce s, ei he cul u ed in op imized condi ions o solu e p oduc ion o me abolically enginee ed o hyd oxyec- oine o e p oduc ion (4). Table 2 summa izes mos o he so- a - epo ed hyd oxyec oine p oduc ion sys ems, including ele an TABLE 1 Ec oine and hyd oxyec oine yield and p oduc ion a es o C. salexigens assayed s ains a S ain Temp (°C) Salini y (%) G ow h a e (␮h ⫺1 ) Ec oine Hyd oxyec oine Yield (␮mol/g BDM) Speci ic p oduc ion a e (␮mol/g BDM · h) b Yield (␮mol/g BDM) Speci ic p oduc ion a e (␮mol/g BDM · h) C. salexigens DSM3043 wild ype 37 4.35 0.24 393 ⫾18.5 94.3 59 ⫾2.9 14.1 37 8.7 0.33 654 ⫾27.6 215.8 202 ⫾7.8 66.6 37 14.5 0.21 725 ⫾22.3 152.2 324 ⫾11.3 68 40 4.35 0.22 180 ⫾8.5 39.6 23 ⫾0.8 5.06 40 8.7 0.30 449 ⫾9.6 134.7 176 ⫾7.8 52.8 45 14.5 0.15 381 ⫾12.2 57.21 942 ⫾20 141 Wild ype/pHYDROX1 37 4.35 0.14 220 ⫾5.4 30.8 483 ⫾9.9 67.62 37 8.7 0.25 156 ⫾3.1 39 424 ⫾7.5 106 40 4.35 0.12 48.5 ⫾1.2 5.8 468 ⫾8.2 56.16 40 8.7 0.24 20.4 ⫾0.4 4.9 255 ⫾3.3 61.2 Wild ype/pHYDROX2 37 4.35 0.18 68 ⫾1.2 12.2 384 ⫾6.8 69.12 37 8.7 0.29 108 ⫾2.4 31.3 361 ⫾7.3 104.6 40 4.35 0.17 48.85 ⫾0.9 8.3 419 ⫾9.3 71.23 40 8.7 0.24 105 ⫾3.4 25.2 354 ⫾10.1 84.96 CHR137/pHYDROX1 37 4.35 0.14 81 ⫾2.2 11.3 598 ⫾12.8 83.72 37 8.7 0.1 170 ⫾4.1 17 632 ⫾14.5 63.2 40 4.35 0.13 36.5 ⫾1.4 4.7 478 ⫾12.2 62.14 40 8.7 0.1 90 ⫾3.3 9 521 ⫾16.4 52.1 CHR137/pHYDROX2 37 4.35 0.14 93 ⫾2.9 13 883 ⫾18.9 123.62 37 8.7 0.1 286 ⫾5.8 28.6 967 ⫾24.1 96.7 40 4.35 0.12 67.5 ⫾3.1 8.1 768 ⫾31.2 92.1 40 8.7 0.11 122.6 ⫾4.5 13.48 805 ⫾25.2 88.5 a Expe imen s we e epea ed wice wi h h ee independen measu emen s. The esul s a e a e ages om he six measu emen s ⫾s anda d de ia ions. b BDM, bac e ial d y ma e . Rod íguez-Moya e al. 1020 aem.asm.o g Applied and En i onmen al Mic obiology on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om TABLE 2 Compa ison o hyd oxyec oine p oduc ion sys ems S ain Temp (°C) Salini y (%) G ow h phase G ow h a e (␮h ⫺1 ) OH-ec oine yield (␮mol/g BDM) Speci ic OH-ec oine p oduc ion a e (␮mol/ gBDM·h) a Byp oduc (s) Reac o sys em P oduc ex ac ion Re e ence Nonhalophilic p oduce s E. coli DH5␣/pSB01 b 37 2.0 La e s a iona y 0.35 500 175 T ehalose ec oine (⬍5%) Ba ch Me hanol-chlo o o m ex ac ion 17 E. coli FF4169/pNST5 c 37 1.74 A e 16 h o incuba ion NR k 29.95 NR Ec oine (10.32 ␮mol/g BDM), o he solu es no es ed Ba ch Me hanol-chlo o o m ex ac ion 19 E. coli FF4169/pNST6 d 37 1.74 A e 16 h o incuba ion NR 91 NR Ec oine (107 ␮mol/g BDM), o he solu es no es ed Ba ch Me hanol-chlo o o m ex ac ion 19 Na u al p oduce s Ma inococcus sp. s ain M52 35 10.0 S a iona y 0.25 860 215 Glu ama e Fed-ba ch-ba ch e Me hanol-chlo o o m ex ac ion 20 Ma inococcus sp. s ain M52 37 10.0 S a iona y 0.20 670 134 Ba ch- ed-ba ch e The mal pe meabiliza ion 21 Ma inococcus sp. s ain M52 37 10.0 S a iona y 0.03 603 18 Ba ch- ed-ba ch e The mal pe meabiliza ion 21 Halomonas boli iensis 35 18.5 S a iona y Mul is ep p ocess 950 169 Ec oine, PHB Two-s ep ed-ba ch Downshocks 22 Halomonas elonga a ATCC 33173 T 25 15 Exponen ial 0.04 28 NR Ec oine (1,462 ␮mol/g BDM), N␥- ace yldiamino-bu y ic acid, glu ama e, alanine, o he amino acids Ba ch- ed-ba ch Downshocks 9 Halomonas elonga a ATCC 33173 T 30 10.0 La e exponen ial 0.242 36 8.71 Ec oine (762 ␮mol/g BDM), glu ama e, glucose Ba ch Chlo o o m ex ac ion 23 Halomonas elonga a ATCC 33173 T 40 15 La e exponen ial NR 290 NR Ec oine (740 ␮mol/g BDM), glu ama e, glucose Ba ch Me hanol-chlo o o m ex ac ion 24 Halomonas elonga a ATCC 33173 T 40 20 La e exponen ial NR 440 NR Ec oine (860 ␮mol/g BDM), glu ama e, glucose Ba ch Me hanol-chlo o o m ex ac ion 24 Ch omohalobac e salexigens DSM 3043 T 37 14.5 Ea ly s a iona y 0.21 324 68 Ec oine (725 ␮mol/g BDM), glu ama e Ba ch Me hanol-chlo o o m ex ac ion 13; his s udy Ch omohalobac e salexigens DSM 3043 T 45 14.5 Ea ly s a iona y 0.15 942 141 Ec oine (381 ␮mol/g BDM), glu ama e, ehalose, N␥- ace yldiamino-bu y ic acid Ba ch Me hanol-chlo o o m ex ac ion 2; his s udy Ch omohalobac e salexigens DSM 3043 T 37 10.75 Exponen ial 0.3 2,528 76 Ec oine (3,797 ␮mol/g BDM), o he solu es no de e mined Con inuous wi h cell e en ion g Downshocks 11 Pseudomonas s u ze i DSM5190 T 37 5.0 Exponen ial 0.16 480 76.8 Ec oine, ehalose, NAGGN Ba ch Me hanol-chlo o o m ex ac ion 17 Pseudomonas s u ze i A1501 37 4.0 Mid-exponen ial NR 367 NR Ec oine, ehalose, NAGGN Ba ch Me hanol-chlo o o m ex ac ion 19 Enginee ed na u al p oduce s C. salexigens DSM 3043 T /pJP-2R h 37 10 Ea ly s a iona y NR 100 i NR 0% ec oine, o he solu es no de e mined Ba ch Downshocks 25 C. salexigens DSM 3043 T /pHYDROX2 j 37 4.35 Ea ly s a iona y 0.18 384 69.12 Glu ama e, ehalose, glycolsylglyce ol, ec oine Ba ch Me hanol-chlo o o m ex ac ion This s udy C. salexigens CHR137/pHYDROX2 j 37 4.35 Ea ly s a iona y 0.14 883 123.62 Glu ama e, ehalose, ec oine (93 ␮mol/g BDM) Ba ch Me hanol-chlo o o m ex ac ion This s udy a Maximum speci ic hyd oxyec oine p oduc ion a es we e calcula ed on he basis o g ow h a es and biomass con en . b Ca ying he ec ABCDask genes om P. su ze i DSM 5190 T . c T ehalose-de icien E. coli ca ying he ec ABCD genes om P. s u ze i A1501. d T ehalose-de icien E. coli ca ying he ec ABCDasK genes om P. s u ze i A1501. e Wi h medium exchange once, du ing ed-ba ch. Cells we e i s g own du ing 24 h a op imal salini y o biomass p oduc ion (4.5% NaCl) and hen ans e ed o a high-salini y medium (wi h 18.5% NaCl) o ec oines p oduc ion. g Calcula ions o a maximum biomass o 61 g/li e . Fe men a ion was op imized o a simul aneous p oduc ion o ec oine and hyd oxyec oine. h Ca ying he hpD (ec D) gene, encoding he ec oine hyd oxylase om S ep omyces c ysomallus. i Pe cen age o ec oine con e sion o hyd oxyec oine; absolu e yield no epo ed. j Ca ying he ec ABCDD casse e om C. salexigens DSM 3043 T . k NR, no epo ed. O e p oduc ion o Hyd oxyec oine by Ch omohalobac e Feb ua y 2013 Volume 79 Numbe 3 aem.asm.o g 1021 on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om pa ame e s such as g ow h condi ions, hyd oxyec oine yield and p oduc ion a e, by-p oduc s, eac o sys ems, and p oduc ex- ac ion p ocedu es used. In his wo k, a emp s o o e p oduce he C. salexigens ec ABCD hyd oxyec oine syn hesis genes in E. coli DH5␣we e unsuccess ul. I is possible ha , as obse ed by Bes a e e al. (26) o he he e ologous p oduc ion o he Ma i- nococcus halophilus ec oine syn hesis genes, ou he e ologous p o- duc ion sys em migh be imp o ed by coexp essing a eedback- insensi i e aspa a e kinase. This me abolic bo leneck was no ound in he wo epo s so a desc ibing success ul hyd oxyec- oine p oduc ion by E. coli ca ying he hyd oxyec oine syn hesis genes om Pseudomonas s u ze i. Howe e , di e ences in he os- mo ole ance con e ed o he hos s ain, hyd oxyec oine yield, p esence o by-p oduc s, and he dependence o coexp esion o he ask gene o enhanced ec oine/hyd oxyec oine p oduc ion we e ound (17,19)(Table 2), Thus, he ehalose-de icien E. coli s ain FF4169 bea ing he P. s u ze i A1501 ec ABCD genes accu- mula ed mode a e le els o ec oine and hyd oxyec oine, and he in oduc ion o he ask gene (encoding an aspa a e kinase special- ized o ec oine/hyd oxyec oine syn hesis) led o a e y s ong inc ease in he con en s o bo h solu es (Table 2). In bo h cases, hos cells we e osmop o ec ed (19). In con as , E. coli DH5␣ca - ying he ec ABCDask gene clus e om P. s u ze i DSM 5190 T syn hesized almos exclusi ely hyd oxyec oine as compa ible sol- u e (wi h abou 5% o ec oine and ehalose), al hough con e sion o ec oine in o hyd oxyec oine was delayed un il la e s a iona y phase. Su p isingly, his s ain was no osmo ole an , and en- hanced hyd oxyec oine p oduc ion was no dependen on coex- p ession o he aspa a e kinase (17). Despi e all his, hyd oxyec- oine con en was much highe han ha obse ed in he na u al p oduce , wi h a yield o 500 ␮mol/g BDM (a s a iona y phase) and a speci ic p oduc ion a e o 175 ␮mol/g BDM·ha 37°C and 2% NaCl. Compa ed o his s ain, he speci ic hyd oxyec oine p oduc ion a e o C. salexigens CHR137/pHYDROX2 g own a he same empe a u e wi h 4.35% NaCl was sligh ly lowe [123.6 ␮mol/g BDM · h], bu he hyd oxyec oine yield was much highe (883 ␮mol/g BDM) (Table 2). Fo he indus ial p oduc ion o hyd oxyec oine, obus mi- c oo ganisms wi h a b oad sal ole ance a e a o ed o pe o m he bac e ial milking p ocess (3,4). Me hods based on he G am- posi i e Ma inococcus sp. s ain M52 (20,21) yielded conside able hyd oxyec oine amoun s a 37°C (Table 2) bu had wo disad an- ages: (i) hey canno be milked by simple dilu ion o he medium, and (ii) hey p oduce g ow h-inhibi ing componen s such as ac- e a e, which impede hei use in ba ch and ba ch e men a ion p ocesses, unless complex echniques a e u ilized. Among G am- nega i e bac e ia o he Halomonadaceae amily, C. salexigens (2), H. boli iensis (22), and H. elonga a (9,24) na u ally p oduce mo e ec oine han hyd oxyec oine, and inc easing he hyd oxyec oine con en implies high- empe a u e and -salini y g ow h condi ions (Table 2), wi h he disad an ages ha hese ex eme condi ions ha e o any indus ial p oduc ion sys em. In con as , in P. s u ze i, hyd oxyec oine is he p edominan compa ible solu e a no mal empe a u e (Table 2), and his mic oo ganism has been sugges ed as an in e es ing candida e o he bio echnological p o- duc ion o hyd oxyec oine (19). A p esen , hyd oxyec oine is p oduced on an indus ial scale wi h H. elonga a ATCC 33173 T (same as H. elonga a DSM 2581 T ). Bo h C. salexigens DSM 3043 T (s ain 1H11; o me ly named H. elonga a DSM 3043) and H. elonga a ATCC 33173 T (s ain1H9) we e isola ed by V eeland e al. om a sola sal acili y a Bonai e Island and ini ially assigned o H. elonga a (27). On he basis o hei pheno ypic di e ences and hei phylogene ic dis ance, s ain H. elonga a DSM 3043 was p oposed as a new species o he genus Ch omohalobac e and designa ed C. salexigens (7). Al- hough bo h species show a simila empe a u e ange ( om 15 o 45°C, wi h op imum a 37°C) and op imal salini y (8.7 o 11.6% NaCl) o g ow h (28,29), C. salexigens DSM 3043 seems o ha e mo e s ingen equi emen s o sal . Thus, while he H. elonga a ype s ain g ew well wi h 0.3% NaCl in a minimal medium which is simila o M63 (29), C. salexigens could no g ow a all in M63 unless i con ained 2.9% NaCl (28). The e a e ew epo s desc ib- ing hyd oxyec oine p oduc ion by H. elonga a. Ea ly s udies by Wohl a h e al. (24) and Se e in e al. (23) showed inc easing hyd oxyec oine con en in H. elonga a in esponse o salini y and empe a u e, wi h 290 ␮mol/g BDM o hyd oxyec oine (and 740 ␮mol/g BDM o ec oine) in cells g own in glucose mine al me- dium a 40°C wi h 15% NaCl (Table 2). This yield is lowe han he hyd oxyec oine accumula ed by he C. salexigens wild- ype s ain a 37 o 45°C wi h a simila salini y (Table 2). Un o una ely, hyd oxyec oine p oduc ion by H. elonga a a empe a u es highe han 40°C o speci ic hyd oxyec oine p oduc ion a es a 40°C o highe we e no epo ed, making i di icul o compa e p oduc- ion da a among he wo wild- ype s ains. In his s udy, we desc ibe hyd oxyec oine o e p oduc ion by using ecombinan s ains o he na u al p oduce C. salexigens gene ically enginee ed (i) o coexp ess, in a na i e-plasmid-based ec o , he ec oine and hyd oxyec oine genes unde he con ol o he ec A p omo e egion, (ii) o imp o e he i s app oach by inc easing he copies o ec D, and (iii) o u he imp o e he wo i s designs by using an ec oine-de icien mu an as he gene ic backg ound. As s a ed abo e, ansc ip ional egula ion o he C. salexigens ec ABC genes o ec oine syn hesis is a he complex, wi h a o al o ou p omo e s egula ing ec ABC ansc ip ion ( wo pu a i e ␴ 70 -dependen p omo e s, one ␴ S -con olled p o- mo e , and a ou h p omo e o unknown speci ici y) and one pu a i e ␴ 32 -dependen p omo e d i ing ec BC exp ession. This mul iplici y o p omo e s allows he cells o espond o many en- i onmen al s imuli such as high salini y and empe a u e and he p esence o i on, ex e nal osmop o ec an s, o he DNA gy ase inhibi o nalidixic acid (13). In H. elonga a ATCC 33173 T , Schwibbe e al. (30) ound a di e en bu also complex p omo e assembly, wi h wo ansc ip ional ini ia ion si es ups eam o ec A (co esponding o pu a i e ␴ 70 - and ␴ S -dependen p omo - e s), and a hi d one mapped immedia ely ups eam o ec C, e- sembling ␴ 54 -con olled p omo e s. Based on hese indings, he au ho s sugges ed ha ec oine syn hesis in H. elonga a could be egula ed no only by salini y bu also by ni ogen supply. In ad- di ion, in he p esence o he ex e nal osmop o ec an be aine, ec oine accumula ion is o ally abolished in C. salexigens (28) bu no in H. elonga a (24), e lec ing an appa en ly di e en egula- ion o ec oine p oduc ion in bo h o ganisms. In summa y, wi h he a ailable da a, i is di icul o p edic i he same s a egy ollowed in his s udy would e icien ly wo k in H. elonga a. As s a ed abo e, he p esence o by-p oduc s in any s ain de- o ed o indus ial p oduc ion o hyd oxyec oine is undesi able, as i would inc ease p oduc ion cos s (4). All s ains depic ed in Table 2 ha ha e been es ed o he p esence o o he solu es show om ace (i.e., E. coli DH5␣/pSB01) o mode a e (i.e., H. elonga a,C. salexigens,o P. s u ze i wild- ype s ains) amoun s o Rod íguez-Moya e al. 1022 aem.asm.o g Applied and En i onmen al Mic obiology on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om o he con amina ing solu es. The e o e, downs eam p ocesses in ol ing a ce ain pu i ica ion s ep a e mos ly una oidable. Fo he mos p omising s ain, CHR137/pHYDROX2, hyd oxyec o- ine was he main p oduc , which would be use ul o educe sepa- a ion and pu i ica ion cos s. In any case, sepa a ion o hy- d oxyec oine om ehalose and glu ama e is easy by simple ch oma og aphic echniques (3,9). Ou indings indica e he supe io i y o he C. salexigens ec o- ine-minus s ains o e he wild- ype s ain o hyd oxyec oine p oduc ion, including much be e yields and speci ic p oduc ion a es han he wild ype. P oduc ion achie ed wi h s ain CHR137/pHYDROX2 g owing a a ela i ely low salini y, 4.35% NaCl, exceeds ha o mos o he s ains epo ed elsewhe e (Table 2). I s speci ic hyd oxyec oine p oduc ion yield is o he same o de as ha o he e ologous p oduc ion by E. coli DH5␣/pSB01 a 2% NaCl (17), and he absolu e yield is much highe . In addi- ion, con e sion o ec oine o hyd oxyec oine by E. coli/pSB01 was much delayed un il la e s a iona y phase, whe eas in C. salexigens p oduc ion was maximal a ea ly s a iona y phase. Ne e heless, E. coli DH5␣/pSB01 has he ad an age o yielding pu e hy- d oxyec oine. In addi ion, due o i s na u al abili y o cope wi h s ong changes in medium osmola i y, C. salexigens is a much mo e obus s ain han E. coli o he indus ial bac e ial milking p ocess, and he e o e he e is much oom o imp o emen o hyd oxyec oine p oduc ion, o ins ance by con inuous e men- a ion coupled o p oduc ex ac ion by osmo ic downshocks. 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O e p oduc ion o Hyd oxyec oine by Ch omohalobac e Feb ua y 2013 Volume 79 Numbe 3 aem.asm.o g 1023 on Ma ch 3, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://aem.asm.o g/Downloaded om