View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by idUS. Depósito de Investigación Universidad de Sevilla Temperature- and Salinity-Decoupled Overproduction of Hydroxyectoine by Chromohalobacter salexigens Javier Rodríguez-Moya,a Montserrat Argandoña,a Fernando Iglesias-Guerra,b Joaquín J. Nieto,a Carmen Vargasa Department of Microbiology and Parasitologya and Department of Organic and Pharmaceutical Chemistry,b Faculty of Pharmacy, University of Seville, Seville, Spain Downloaded from Hydroxyectoine overproduction by the natural producer Chromohalobacter salexigens is presented in this study. Genetically engineered strains were constructed that at low salinity coexpressed, in a vector derived from a native plasmid, the ectoine (ectABC) and hydroxyectoine (ectD) genes under the control of the ectA promoter, in a temperature-independent manner. Hy- droxyectoine production was further improved by increasing the copies of ectD and using a C. salexigens genetic background unable to synthesize ectoines. ctoine and hydroxyectoine (ectoines) are compatible solutes imal during stationary phase, the accumulation of hydroxyectoine http://aem.asm.org/ Esynthesized and accumulated by halophilic and halotolerant is upregulated by salinity and temperature, and the accumulation bacteria in response to osmotic and heat stress (1, 2). Ectoines of ectoine is upregulated by salinity and downregulated by tem- have current applications as biostabilizers of proteins and nucleic perature. Thus, hydroxyectoine production and accumulation is acids, as well as a potential role as therapeutics for certain diseases maximum at 45°C and 14.5% NaCl, while ectoine accumulation (3, 4). This, together with the complexity of their chemical syn- reaches its maximum at 37°C and 17.4% NaCl (2). This regulation thesis, has encouraged recent efforts to improve ectoine produc- occurs, at least in part, at the transcriptional level. The ectoine tion from bacteria. Hydroxyectoine is especially interesting, as it synthesis genes ectABC can be expressed from two promoter re- seems to confer additional protections derived from its hydroxy- gions, one located upstream of ectA and composed of four puta- on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIA lated nature (3, 4). Ectoines are synthesized from aspartate semi- tive promoters (PectA1 to PectA4 [PectA1-4]) and a second inter- aldehyde. First, this metabolite is converted into diaminobutyric nal promoter located upstream of ectB (PectB). In silico analysis of acid, which is acetylated to N␥-acetyldiaminobutyric acid and the Ϫ10 and Ϫ35 sequences of these regions showed that PectA1 subsequently cycled to ectoine (5, 6). The main route of hy- and PectA2 may be dependent on the main vegetative factor ␴70 droxyectoine synthesis is via ectoine hydroxylation (2). The en- (and therefore constitutively expressed), whereas PectA3 and zymes for ectoine synthesis are usually encoded in an ectABC-type PectB were similar to ␴S- and ␴32-dependent promoters, respec- gene cluster, which is usually well conserved among ectoine-pro- tively. In agreement with these predictions, expression of a ducing microorganisms (7). There are exceptions, such as incom- PectA1-4::lacZ fusion was osmoregulated and depended in part on plete operons, gene clusters including the ask gene (for the aspar- the general stress factor ␴S, whereas PectB was induced by contin- tate kinase), gene clusters carrying ectABC-ectD-ask, and uous growth at a high temperature (13). On the other hand, the scattering of the genes within the chromosome, with duplications promoter region of ectD is composed of two promoters (PectD1 of ectC and ectD or even solitary ectC (5, 7, 8). Industrial produc- and PectD2), and ectD expression is both osmo- and thermoregu- tion of hydroxyectoine uses Halomonas elongata ATCC 33173T lated (M. Reina-Bueno, unpublished data). grown under high-salinity and high-temperature conditions, us- In this work, we have metabolically engineered C. salexigens to ing the bacterial milking method (9), or a derivative of this tech- overproduce hydroxyectoine at low salinity, in a temperature-in- nique (10), followed by separation and purification of ectoine and dependent manner. In order to maximize hydroxyectoine pro- hydroxyectoine (9). This salt and temperature requirement for duction and to minimize its temperature and salinity require- hydroxyectoine synthesis is a serious drawback of using natural ments, we designed transcriptional fusions between the ectoine producers, since fermentation under high temperature and salin- synthesis genes ectABC and the main hydroxyectoine synthesis ity increases production costs and corrodes industrial reactors. gene ectD, so that the second became transcriptionally controlled Chromohalobacter salexigens is a halophilic gammaproteobac- by the ectABC promoter region. To construct a functional terium which produces ectoine and hydroxyectoine in response to ectABCD cassette, we first amplified by PCR a 3,384-bp sequence, salt and heat stress, respectively (7). It is easy to grow and its including the promoter region upstream of ectA, the ectABC gene genome sequence is available (http://genome.ornl.gov/microbial cluster, and the rho-independent terminator downstream of ectC, /csal/). It has been suggested as an alternative to H. elongata (11). In C. salexigens, the genes encoding ectoine synthesis lay within a 2.8-kb region encoding the diaminobutyric acid acetyltransferase Received 8 September 2012 Accepted 9 November 2012 (EctA), diaminobutyric acid transaminase (EctB), and ectoine Published ahead of print 16 November 2012 synthase (EctC) (12). The microorganism has two paralogs of the Address correspondence to Carmen Vargas, [email protected]. enzyme ectoine hydroxylase, EctD and EctE, but EctD is the main Supplemental material for this article may be found at http://dx.doi.org/10.1128 responsible enzyme for hydroxyectoine production (2). In C. /AEM.02774-12. salexigens, the gene cluster ectABC and the genes ectD and ectE are Copyright © 2013, American Society for Microbiology. All Rights Reserved. at different loci within the chromosome. doi:10.1128/AEM.02774-12 Whereas accumulation of both solutes in C. salexigens is max- 1018 aem.asm.org Applied and Environmental Microbiology p. 1018–1023 February 2013 Volume 79 Number 3 Overproduction of Hydroxyectoine by Chromohalobacter FIG 1 Genetic organization of constructed inserts, cloned in pHS15 to obtain pHYDROX1 (A) and pHYDROX2 (B). Downloaded from and cloned it into pBluescript SK, resulting in pME2. Then, we uid chromatography-mass spectrometry (LC-MS) and superna- inserted a BamHI restriction site between ectC and the rho-inde- tants used for high-pressure liquid chromatography with UV de- pendent terminator by site-directed mutagenesis using the primer tector (HPLC-UV) analysis of ectoine and hydroxyectoine were pair ectBam_fw and ectBam_rv (see Table S1 in the supplemental prepared by using a modified Bligh-Dyer technique described by material for a list of primers used in this study). Subsequently, we Kraegeloh and Kunte (15). For cellular extracts, chromatographic http://aem.asm.org/ eliminated the BamHI restriction site from the multicloning separation and HPLC-electrospray ionization was performed as site of pBluescript SK using the primers QuitBam_fw and described by Argandoña et al. (16). For supernatants, samples QuitBam_rv, getting pME2.3. Next, we amplified a 1,200-bp were analyzed as described by García-Estepa et al. (2). sequence from the C. salexigens genome, including ectD, and Hydroxyectoine levels observed in the heterologous recombi- inserted it into pBluescript SK, getting the plasmid pECTD. nant strains E. coli DH5␣/pHYDROX1 and E. coli DH5␣/ Then, we introduced a BclI restriction site downstream of ectD pHYDROX2 grown at 1%, 2%, or 3% NaCl were very low, and using the primers ectDBcl_fw and ectDBcl_rv, resulting in only traces of ectoine were detected (data not shown). Table 1 pECTD2. Subsequently, we excised promoterless ectD from summarizes growth rates and ectoine and hydroxyectoine pro- pECTD2 by digesting it with BclI and inserted it in BamHI- duction by the different C. salexigens wild-type and recombinant on March 3, 2016 by USE/BCTA.GEN UNIVERSITARIA digested pME2.3, yielding pECTABCD. Finally, the engineered strains, as well as their specific production rates. As production of ectABCD gene cluster was excised from pECTABCD by diges- ectoines is directly related to the biomass produced at a certain tion with EcoRI and cloned into EcoRI-digested pHS15 (a salinity (11), the specific production rate (␮mol/g bacterial dry cloning and expression vector based on a native plasmid from matter [BDM] · h) is a simple function reflecting solute content H. elongata harboring a streptomycin resistance gene [14]), and growth rate (17). As previously reported (2), ectoine accumu- obtaining pHYDROX1 (Fig. 1A). To increase the ectD gene lation by C. salexigens wild type was salinity dependent and, at a dose, a second functional cassette, ectABCDD, was constructed. given salinity, inversely correlated to increasing temperature. For this purpose, a second copy of ectD was cloned between Ectoine reached its maximal accumulation (725 ␮mol/g BDM) at ectD and the rho-independent terminator in the previous ect- 37°C with 14.5% of NaCl, with an ectoine/hydroxyectoine ratio of ABCD-constructed gene cluster. First, we introduced a BamHI 2.24:1. On the other hand, hydroxyectoine accumulation by the restriction site between ectD and