RESUMEN
OBJECTIVE: To construct a clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system and use this system to obtain a recombinant Escherichia coli strain possessing the fatty acid metabolism genes from a lipid-rich marine bacterium. RESULTS: The fatty acid regulatory transcription factor (fadR), delta9 (Δ(9) desaturase) and acetyl-CoA carboxylase (acc) genes were cloned from Shewanella frigidimarina. The fatty acid regulatory transcription factor (fadD) and phosphoenolpyruvate carboxylase inactivated strains were used to construct the fadR/delta9 and acc knock-in strains, which are both markerless and "scar"-less, and identified the change in fatty acid composition in the recombinant strains. There was no change in fatty acid composition between the wild-type strain and recombinant strains. All strains had 11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:1, 17:0 and 18:0 fatty acids, with 16:0 and 18:0 fatty acids being dominant. The total lipid content of each recombinant strain was higher than the wild-type strain, with a maximum of 13.1 %, nearly 5.3 % higher than wild-type strain. CONCLUSION: The CRISPR/cas9 system, in conjunction with λ-Red recombinases, can rapidly and efficiently edit the E. coli genome. The CRISPR/cas9 recombineering machinery can be modified to select biotechnologically-relevant bacteria other than E. coli.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Ácidos Grasos/metabolismo , Recombinasas/metabolismo , Shewanella/genética , Proteínas Bacterianas/genética , Bacteriófago lambda/enzimología , Bacteriófago lambda/genética , Sistemas CRISPR-Cas , Clonación Molecular , Ácidos Grasos/aislamiento & purificación , Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Shewanella/metabolismoRESUMEN
Aureochrome-1 (AUREO1) is a transcription factor that is induced by blue light and controls branching of Vaucheria frigida. We have cloned the gene, NgAUREO1, coding for AUREO1 from Nannochloropsis gaditana, and report that the lipid content in recombinant Saccharomyces cerevisiae was 1.6-fold more than in wild-type S. cerevisiae (6.3 % lipid increased to 10 %). Over-expression of AUREO1 in S. cerevisiae up-regulated the expression of acetyl-CoA carboxylase and acyl-CoA:diacylglycerol acyl-transferase but down-regulated the expression of long-chain-acyl CoA synthetase. This enhanced the accumulation of lipid. This study highlights a novel function of AUREO1 and allows a better understanding of the regulation mechanism of fatty acid metabolism.
Asunto(s)
Metabolismo de los Lípidos , Lípidos/análisis , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Estramenopilos/genética , Factores de Transcripción/metabolismo , Acetil-CoA Carboxilasa/biosíntesis , Coenzima A Ligasas/biosíntesis , Diacilglicerol O-Acetiltransferasa/biosíntesis , Expresión Génica , Perfilación de la Expresión Génica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
A novel cDNA gene, NgLACS, that encodes a long-chain acyl-CoA sythetase (LACS), was cloned from Nannochloropsis gaditana and characterized. The cDNA was 2,360 bp in length, consisting of an ORF of 1,950 bp, a 5'-untranslated region of 88 bp and a 3'-untranslated region of 322 bp. The deduced amino acid sequence of LACS was 649 amino acid residues in length with a predicted molecular weight of 71 kDa and an isoelectric point of pH 7.8. When the alga was treated with excessive nitrogen and iron, and at 15 °C, the proportion of long-chain polyunsaturated acyl-CoAs in the total acyl-CoAs and the abundance of NgLACS cDNA gene transcript were up-regulated. Over-expression of NgLACS in Saccharomyces cerevisiae caused the accumulation of eicosapentaenoic acid and docosahexaenoic acid.
