Study On Metabolic Pathway Of Carotenoid Production In Rhodococcus

Carotenoids are kinds of four terpene organic chemistry pigments and ubiquitous in nature, they have important functions in microorganisms, plants and animals. Microorganisms, and plants can use isoprenes as raw material for the production of carotenoids. The special unsaturated structure of the carotenoid gives its special functions in organism. Beta carotene is involved in the energy transfer process of photosynthesis in plants and cyanobacteria, and in higher animals, carotenoids are a kind of highly efficient free radical quenching agent. Beseides, carotenoids are involved in the resistance of bacteria to the environment in many bacteria. Human beings are closely related to carotenoids, too. Beta carotene is the precursor of vitamin A, participate in a variety of human essential response, lack of vitamin A can cause night blindness. The functions of carotenoids mainly show that it can quench the oxygen free radical, antioxidation, strengthen the immunity of human body and it prevents people from cardiovascular disease, cancer and so on. Under this background, the industrial prospect of producing carotenoids is even brighter. Microbial fermentation is the best healthy and feasible way to product carotenoids under the current circumstance and thus, production of carotenoids by genetic engineering has become more and more bright.As a kind of actinomycetes, Rhodococcus are closely related to human beings and of great significance in the pharmaceutical industry. Rhodococcus can produce kinds of enzyme preparations, vitamins, and organic acids and it has the natural decontamination ability as well. Recent studies have showed that the vast majority of the Rhodococcus produce carotenoids, which makes it possible to produce carotenoids by expressing carotenoid genes of Rhodococcus in vitro. During the initial stage of our experiments, carotenoid producing bacterium Rhodococcus sp. B7740 was found in bacteria isolated form seawater samples collected in the Arctic Ocean. Through a series of primary identification, we comfirmed its ability to produce carotenoids. On the basis of this, studies on expression of carotenoid metabolism genes form Rhodococcus B7740, exploring the metabolic pathway of carotenoid production, construction of carotenoid engineering bacteria have been implemented.1. Whole genome sequencing of Rhodococcus B7740 has been finished in this study and its genome annotation has been accomplished. The genome size of Rhodococcus B7740 was 5341557 bp, with a GC content of 64.93%. Whole genome sequenc of Rhodococcus B7740 contains 5196 predicted coding sequence. There are 47 tRNAs, 4 5S rRNAs, 4 LSU rRNAs, 4 SSU rRNAs, and 424 subsystems represent in the genome. The complete genome sequence of Rhodococcus sp. Strain B7740 is available in GenBank and the accession number is CP010797.2. Rhodococcus B7740 containing carotenoid producing key genes : phytoene synthase gene(Crt B), geranyl geranyl pyrophosphate synthetase gene(Crt E),phytoene dehydrogenase gene(Crt I). These genes mediate the production of lycopene. The serial number of these genes in GenBank is: gb|CP010797.1|.3. This experiment chooses PET30 a plasmid as the carrier frame, and by using overlapping PCR technology, PCR amplification after DNA restriction enzyme digestion and T4 DNA enzyme linked, we constructed Vector PET30a-EBI. Vector PET30a-EBI contains Crt E, Crt B, Crt I genes of Rhodococcus sp. Strain B7740, and we completed co expression of Crt E, Crt B, Crt I three genes in E-coli. However, the results indicate that the co expression of Crt E, Crt B, Crt I three genes in Escherichia coli can not promote the production of lycopene.4. In order to further explore the metabolic pathway of carotenoid production in Rhodococcus, 4 strains of Rhodococcus were selected in this study. These four strains of Rhodococcus were isolated from the sea water samples from the East Pacific Ocean and there are obvious differences in the color of their colonies. We identified the basic characteristics of the carotenoid producing species in this study. Then, draft genome sequencing of this four Rhodococcus species were determined. After the genome annotation, genome sequence were submitted to the Genbank database under the genome BioProject accession number PRJNA314738, version XXXX01000000. Through the analysis of its genome, the carotenoids producing key genes of 4 strains of Rhodococcus were found. The 4 strains of Rhodococcus carotenoids producing genes compared with Rhodococcus B7740, according to the type of pigment production and we find that Crt E, Crt B, Crt I three genes of Rhodococcus sp. EPR-147,Rhodococcus sp. EPR-279,Rhodococcus sp.B7740 are relatively conservative and protein sequence homology are relatively higher. Correspondingly, the colony color of these bacteria are deeper, and their pigment production are relatively higher. In contrast, in comparison with other genes, Crt E, Crt B, Crt I three genes of Rhodococcus sp. EPR-134 are more open and with a low protein sequence homology and Rhodococcus sp. EPR-134 does not produce carotenoids. Analysis of Crt E, Crt B, Crt I three genes of Rhodococcus sp. EPR-157, the result of it seems to ‘between this two kinds’. Genetic changes of Crt E, Crt B, Crt I three genes of Rhodococcus sp. EPR-157 are smaller and Rhodococcus sp. EPR-157 pruduces a small amount of carotenoids.