Increased -Carotene Production in Recombinant Escherichia coli Harboring an Engineered Isoprenoid Precursor Pathway with Mevalonate Addition Sang-Hwal Yoon, ²,‡ Hye-Min Park, ²,‡ Ju-Eun Kim, ² Sook-Hee Lee, ² Myung-Suk Choi, §,| Jae-Yean Kim, ²,§ Deok-Kun Oh, ⊥ Jay D. Keasling, ¶ and Seon-Won Kim* ,²,§ Division of Applied Life Science (BK21), EB-NCRC and PMBBRC, and Division of Forest Science, Gyeongsang National University, Jinju 660-701, Korea, Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-503, Korea, and Department of Chemical Engineering, University of California, Berkeley, California 94720-1462 When pT-LYCm4 containing lycopene synthetic genes was co-transformed with pSUcrtY or pSHcrtY containing crtY gene of Pantoea ananatis (P. ananatis) or Pantoea agglomerans (P. agglomerans), -carotene productions of 36 and 35 mg/L were obtained, respectively. No lycopene was detected in the -carotene production culture. pT-HB, constructed by addition of P. ananatis crtY gene into pT-LYCm4, was used for co-transformation with pSdxs and pSSN12Didi, which increased isopentenyl diphosphate and dimethylallyl diphosphate synthesis. -Carotene production significantly increased 1.5-fold (51 mg/L) with the amplification of the dxs gene through pSdxs and 4-fold (135 mg/L) with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT-DHB, constructed by integrating the dxs gene into pT-HB, was used for cotransformation of Escherichia coli (E. coli) harboring pSSN12Didi, resulting in -carotene production of 141 mg/L. Recombinant E. coli harboring pT-DHB and pSSN12Didi was used to maximize -carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, -carotene production of 503 mg/L in concentration and 49.3 mg/g DCW in content was obtained at 144 h, which was the highest level of carotenoid production in E. coli ever reported in the literature. Introduction Carotenoids are a diverse class of C 40 isoprenoids with multiple physiological and nutritional roles in plants, algae, bacteria, and fungi (1-3). Several carotenoids such as lycopene, -carotene, zeaxanthin, and astaxanthin are important industri- ally as nutrient supplements, food colorants, and feed additives. Scientific interest in dietary carotenoids has increased in recent years due to their beneficial effects on human health, such as a reduced risk of cancer and enhancement of immune system function (3), which are attributed to their antioxidative potential. Carotenoids used in industry are mostly manufactured using chemical synthesis or are natural extracts or concentrates. The increasing interest in microbial carotenoid sources is related to consumer preferences for natural additives and the potential cost effectiveness of creating carotenoids via microbial biotechnology (4). In the past few decades, progress has been made within the field of biosynthesis of carotenoid in bacteria, fungi, and plants (2, 5). Lycopene, zeaxanthin, and astaxanthin have been successfully synthesized in non-carotenogenic Escherichia coli (E. coli)(6-8). However, little progress has been made in producing -carotene in E. coli using metabolic engineering. Of all known carotenoids, -carotene is believed to be the most important in human nutrition. Animals, in general, are unable to synthesize retinoids (vitamin A and its derivatives) de novo and rely on a dietary supply of these compounds in the form of provitamin A carotenoid, -carotene from plants (9). Retinoids are essential components for vision and are also essential in the maintenance of normal growth and development, immunity, and reproduction (10). The coexpression of four exogenous genes in E. coli cellss GGPP synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI), and lycopene cyclase (crtY)sis sufficient for the conversion of isopentenyl diphosphate (IPP) and farnesyl diphosphate (FPP) to a yellow-colored -carotene (Figure 1) (11). High-yield production of carotenoids in engineered microbial hosts requires optimizing the availability of isoprenoid precursor pool of IPP and DMAPP (dimethylallyl diphosphate) and balancing the expression of carotenogenic genes for efficient transformation of the precursors to the desired carotenoid compounds. Two pathways for the synthesis of IPP and DMAPP exist: the well-known mevalonate pathway and the newly discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (12). Eukaryotes usually use the mevalonate pathway exclusively to convert acetyl-CoA to IPP, which is subsequently isomerized to DMAPP. Prokaryotes, with some exceptions (13), use the MEP pathway to produce IPP and DMAPP separately through a branch point (14). Plants and Streptomycetes use both pathways (15, 16). The MEP pathway has been engineered to * Corresponding author. Telephone: +82-55-751-6522. Fax: 82-55-759- 9363. E-mail: swkim@gsnu.ac.kr. ² Division of Applied Life Science (BK21), Gyeongsang National University. ‡ These authors contributed equally to this work. § EB-NCRC and PMBBRC, Gyeongsang National University. | Division of Forest Science, Gyeongsang National University. ⊥ Konkuk University. ¶ University of California, Berkeley. 599 Biotechnol. Prog. 2007, 23, 599-605 10.1021/bp070012p CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 05/15/2007