APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Engineering the halophilic bacterium Halomonas elongata to produce β-carotene Marta Rodríguez-Sáiz & Cristina Sánchez-Porro & Juan Luis De La Fuente & Encarnación Mellado & José Luis Barredo Received: 28 June 2007 / Revised: 5 September 2007 / Accepted: 6 September 2007 / Published online: 27 September 2007 # Springer-Verlag 2007 Abstract Engineering halophilic bacteria to produce car- otenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with β-carotene the most prominent. With this target, we expressed the β-carotene biosynthetic genes crtE, crtY , crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure β-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY , crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of β-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 μg per g of dry weight) in 2% NaCl. On the contrary, no β-carotene was detected in 15% NaCl. Successful construction of the β-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production. Keywords Carotene . Halophilic . Halomonas . Engineering . Carotenoid Introduction Mainly two physiological groups inhabit saline and hypersaline environments: the extremely halophilic and the moderately halophilic bacteria and archaea (Ventosa et al. 1998). Moderate halophiles comprise a heterogeneous group of species including some methanogenic archaea and a large number of bacteria (Ventosa et al. 1998). Among the moderate halophiles, the genus Halomonas groups a great number of species that can grow in an extended range of salinities (1–30% NaCl), presenting the advantage to be useful in biotechnological processes running at different salinities. The genus Halomonas was initially proposed by Vreeland et al. (1980) with a single species, Halomonas elongata, which included some moderately halophilic bacteria isolated from a saltern located on the Netherlands Antilles. H. elongata ATCC 33173 T constitute the type strain of the genus and, because it is able to grow over a wide range of salinities, it has been used as a model for the study of osmoregulatory mechanisms in halophilic bacteria (Ventosa et al. 1998). Saltern ponds are frequently colored due to the presence of the β-carotene-rich alga Dunaliella salina (Ben-Amotz and Avron 1990), to C 50 -carotenoids including bacterioruberin (Straub 1987) produced by halophilic Archaea, or to the C 40 -carotenoid acyl glycoside produced by the extremely halophilic bacterium Salinibacter ruber (Lutnaes et al. 2002). The pigments produced by these halophilic organ- isms comprise phytoene, β-carotene, lycopene, and deriva- tives of bacterioruberin. Nevertheless, there is no description of halophilic bacteria producing β-carotene as the main carotenoid. Likewise, because carotenoids are not synthe- sized de novo in animals, it seems reasonable to envisage a bacterial origin, at least partially, of carotenoid pigments found in deep-sea shrimps (Nègre-Sadargues et al. 2000). Appl Microbiol Biotechnol (2007) 77:637–643 DOI 10.1007/s00253-007-1195-2 M. Rodríguez-Sáiz : J. L. De La Fuente : J. L. Barredo (*) R&D Biology, Antibióticos S. A., Avenida de Antibióticos 59-61, 24009 León, Spain e-mail: JBarredo@antibioticos.it C. Sánchez-Porro : E. Mellado Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, C/ Profesor García González n° 2, 41012 Sevilla, Spain