Recombinant production of omega-3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1
Recombinant production of omega-3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1
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DOI：
10.1111/j.1365-2672.2010.04817.x
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发表时间：
2010-12-01
影响因子：
4
通讯作者：
Griffiths, M. W.
中科院分区：
文献类型：
--
作者：
Amiri-Jami, M.;Griffiths, M. W.
目的：从波罗的海希瓦氏菌（Shewanella baltica）MAC 1中分离二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）基因，并在E.方法和结果：从S. coli的基因组DNA中构建了一个fosmid文库。Baltica MAC 1的部分片段进行菌落杂交，筛选EPA和DHA基因。波罗的海MAC 1 pfaA和pfaD基因作为探针。从转基因大肠杆菌中分离的总脂肪酸分析。大肠杆菌pfaA和pfaD基因阳性的气相色谱和气相色谱-质谱分析表明，EPA和DHA的重组生产。对分离的基因簇的完整核苷酸序列的分析显示16个推定的开放阅读框（ORF）。其中4个开放阅读框与EPA和DHA生物合成基因簇的pfaA、pfaB、pfaC和pfaD基因同源，但蛋白质结构域与其它EPA/DHA生物合成基因不同。转基因E.携带omega-3基因簇的大肠杆菌菌株能够产生EPA和DHA。分离的基因簇包含在E.这项研究的意义和影响：这些发现对未来在其他微生物中使用EPA和DHA基因簇具有影响，特别是那些用于发酵的微生物。利用E.大肠杆菌或任何其他微生物都具有为各种工农业产品增加经济价值的巨大潜力。
Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost-effective, sustainable and convenient alternative sources for fish oils.Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography-mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes.Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega-3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli.Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro-organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro-organism has great potential to add economic value to a variety of industrial and agricultural products.