Isolation Of EPA/DHA Producing Arctic Bacteria And Study Of Their Polyketide Synthase Pathway

Omega-3 very long-chain polyunsaturated fatty acids(ω-3VLCPUFAs) including eicosapentaenoic acid(EPA, C22:5) and docosahexaenoic acid(DHA, C22:6) play crucial physiological roles in human health.Currently the main source of these nutritionally and medically important fatty acids is fish oil. However, fish oils originated ω-3 VLCPUFAs are insufficient to meet market demand due to increasing contamination and limited productivity.ω-3 VLCPUFAs from algae and marine bacteria as important alternative sources are explored. DHA has been commercially produced from microalgae such as Crypthecodinium cohnii and Schizochytrium sp. But so far, attempts to produce EPA have not yet been successful. In addition, investigations on ω-3 VLCPUFAs from marine bacteria are relatively rare. In this study, EPA-producing stains will be screened from a variety of marine bacteria isolated from Actic areas. Besides, mechanism of DHA production via polyketide synthase pathway in a marine bacterium will be revealed as well.First, prelimilaryly screening of EPA-producing strains from 176 Actic bacteria by using both 2,3,5-triphenyltetrazolium chloride(TTC) color reaction and gas chromatography(GC) methods. Then gas chromatography coupled to mass spectrometry(GC-MS) was used to further characterize the structure of target product. Results showed that four strains could synthesize EPA. Sequencing of 16 S rDNA of these strains suggested they belonged to Nocardioides plantarum, Compostimonas suwonensis, Flavobacterium omnivorum and Shewanella baltica. Of note, it was the first report that EPA production in both Nocardioides and Compostimona species. Additionally, Shewanella baltica 6-42 accumulated up to 4.9% EPA to the total fatty acids. Therefore, effects of temperature and cerulenin on EPA production in strain 6-42 were investigated. When cells were cultured at 0°C, strain 6-42 produced EPA at 8.8% of total fatty acids. It was increased by 1.5 times when compared to that of cells cultured at 10°C. Addition of 3 mg/mL cerulenin could also accelerate EPA content(7.6%) by 1.6 times when cells cultured at 10°C. Moreover, EPA content was further increased up to 11.6% of total fatty acids, that was 2.4 times to the original content(4.9%), when cells cultured at 0°C with supplemented cerulenin.The completion of the 5.1-Mb genome sequencing of strain 6-42 revealed 4597 genes with GC content of 41.67%, involved in life cycle. We focus on pfa gene cluster including five pfa genes responsible for EPA synthesis via Polyketide Synthase(PKS) pathway. Genetic analysis of the pfa gene cluster exihibited that PfaABCD protein had high similarities to those of other Shewanella, while PfaE showed low similarities to those of other strains. Besides, collinearity analysis suggested structural genome of 6-42 was quite diversified from two other stains in the same species, S.baltica OS155 and S.baltica OS678.Second, mechanism of DHA synthesis via PKS pathway in an Arctic isolated Colwellia psychrerythraea 34 H was studied. According to the genome information, pfaEABCD was discovered with around 20 Kb in length.Fragments pfaE and pfaABCD were amplified by using high-fidelity polymerase. Then plasmids containing pfaE or pfaABCD were constructed, named as the pSTV28:: pfaE(DHA) and pDHA△pfaE.Results showed that recombinant DH5a harboring pSTV28:: pfaE(DHA) and pDHA△pfaE produced 3.4% DHA of the total fatty acids at 15°C. In contrast, control cells without pSTV28:: pfaE(DHA) could not accumulate DHA. In addition, four individual pfaE from DHA, EPA or ARA producing strains was synthesized and inserted into pSTV28 to generate pfaE(DHA-M), pfaE(EPA) and pfaE(ARA), respectively. Coexpression of pfaABCD and different pfaE in DH5a made all the recombinant cells produce DHA. Of them, cells harboring pfaE(EPA) and pfaABCD accumulate highest DHA content of 3.7% to the total fatty acids. Furthermore, effects of temperature and cerulenin on the DHA production were disseted as well.Culturing at lower temperature(10°C) was beneficial for recombinant cells to accumulate DHA.The DHA content of recombinant cells harboring pfaE(EPA) and pfaABCD went up to 4.1%, which was increased by 13.7 times compared tothat of cells cultred at 20°C.Results also indicated that addition of cerulenin increased the DHA content.Specifically, the DHA content of recombinant cells harboring pfaE(DHA) and pfaABCD was 7.0%, when addition of 2 mg/mL of ceruleninin to culturing medium.