Date Published: June 2, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Xian Xu, Liqing Tian, Jiali Xu, Chengjia Xie, Ling Jiang, He Huang.
Deinococcus wulumuqiensis R12 is a red-pigmented extremophilic microorganism with powerful antioxidant properties that was isolated from radiation-contaminated soil in Xinjiang Uyghur Autonomous Region of China. The key carotenoid biosynthesis genes, crtE, crtB and crtI, which are related to the cells’ antioxidant defense, were identified in the sequenced genome of R12 and analyzed. In order to improve the carotenoid yield in engineered Escherichia coli, the origin of carotenoid biosynthesis genes was discussed, and a strain containing the R12 carotenoid biosynthesis genes was constructed to produce lycopene, an important intermediate in carotenoid metabolism. The gene order and fermentation conditions, including the culture medium, temperature, and light, were optimized to obtain a genetically engineered strain with a high lycopene production capacity. The highest lycopene content was 688 mg L−1 in strain IEB, which corresponds to a 2.2-fold improvement over the original recombinant strain EBI.
Lycopene is a representative molecule from the carotenoid family, and is one of the strongest antioxidants known to date. Due to its physiological effects (e.g. immune enhancement, free radical scavenging), lycopene is widely used in various fields, such as medicine, food and cosmetics (Moise et al. 2013; Ciriminna et al. 2016). Lycopene production by microbial fermentation has attracted much attention in recent years because of the identification of biosynthetic genes and the discovery of new highly productive pigment-producing strains. The strains that are used to produce lycopene mainly include microbes that can synthesize lycopene naturally, such as Blakeslea trispora, Erwinia herbicola, Rhodotorula genus, or Dunaliella salina, and engineered microbes, such as Escherichia coli, Saccharomyces cerevisiae, Candida utilis, or Yarrowia lipolytica (Hernández-Almanza et al. 2016; Mantzouridou and Tsimidou 2008; Miura et al. 1998). A new species with powerful antioxidant capacity, Deinococcus wulumuqiensis R12, was screened from an irradiated area in Xinjiang province (Wang et al. 2010). It appears red to the unaided eye because of its production of carotenoids, which is one of the major mechanisms of its radiation resistance. Due to this, the radiation-resistant R12 strain can be used as a new platform for carotenoid synthesis, as well as a model for research on the biological adaptations of extremely radioresistant bacteria.
Many efforts have been made to improve the yield of lycopene by engineering bacteria, mostly via the expression of exogenous crtE, crtB and crtI genes for lycopene synthesis from Erwinia to Pantoea species. Yoon et al. constructed engineered E. coli strains harboring lycopene genes from Pantoea agglomerans and Pantoea ananatis, which produced 60 and 35 mg L−1 of lycopene, respectively (Yoon et al. 2007). When the genes crtE, crtB and crtI from Erwinia uredovora were integrated into Candida utilis, it produced a lycopene yield of 758 μg g−1 DCW (Miura et al. 1998). Matthaus et al. constructed a plasmid harboring crtB and crtI from Pantoea ananatis and transformed Yarrowia lipolytica, which produced 16 mg g−1 DCW of lycopene (Matthäus et al. 2014). When the lycopene synthesis genes from different bacteria were cloned into the pGAPZB plasmid and introduced into Pichia pastoris X33, the recombinant strain showed a lycopene production of 73.9 mg L−1 (Bhataya et al. 2009). Bahieldin et al. constructed a plasmid harboring the crt genes from Pantoea ananatis under the control of the ADH2 promoter and introduced it into Saccharomyces cerevisiae, which produced a yield of 3.3 mg lycopene g−1 DCW (Bahieldin et al. 2014). Thus, diverse sources of lycopene synthesis genes expressed in different hosts resulted in different lycopene yields. However, the lycopene synthesis genes from extremophilic radiation-resistant microorganisms were rarely investigated. In this work, the lycopene synthesis genes from the recently isolated extremophilic microorganism Deinococcus wulumuqiensis R12 were analyzed and cloned in E. coli. The transgenic E. coli strain EBI produced a high content of lycopene after twin optimization of fermentation conditions and gene expressing levels (Fig. 9), and thus provides a new microbial gene source for lycopene synthesis and lays a good foundation for improving lycopene production in engineered Escherichia coli.Fig. 9Lycopene production was improved by the combined optimization of culture conditions and gene order. The E. coli strain EBI produced a high content of lycopene after twin optimization of fermentation conditions and gene expressing levels