Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production

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dc.citation.titleBiotechnology for Biofuels
dc.citation.volume287
dc.creatorComba, Santiago
dc.creatorSabatini, Martín
dc.creatorMenendez Bravo, Simón M.
dc.creatorArabolaza, Ana Lorena
dc.creatorGramajo, Hugo Cesar
dc.date.accessioned2024-09-16T13:38:08Z
dc.date.available2024-09-16T13:38:08Z
dc.date.issued2014-12
dc.description.abstractBackground: Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations. Results: The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lppβ genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958 ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L-1 at the end of the process. Conclusions: This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.
dc.description.filFil: Comba, Santiago. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Sabatini, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Menendez Bravo, Simón M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Arabolaza, Ana Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.filFil: Gramajo, Hugo Cesar. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.
dc.description.sponsorshipAgencia Nacional de Promoción Científica y Tecnológica (ANPCyT): PICT 2012-0168, PICT 2011-2005
dc.format.extent1-11
dc.identifier.issn1754-6834
dc.identifier.urihttps://hdl.handle.net/2133/27805
dc.language.isoen
dc.publisherBMC
dc.relation.publisherversionhttps://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-014-0172-0
dc.relation.publisherversionhttps://doi.org/10.1186/s13068-014-0172-0
dc.rightsopenAccess
dc.rights.holderComba, Santiago
dc.rights.holderSabatini, Martín
dc.rights.holderMenendez Bravo, Simón M.
dc.rights.holderArabolaza, Ana Lorena
dc.rights.holderGramajo, Hugo Cesar
dc.rights.holderUniversidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas
dc.rights.textAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectTag biosynthesis
dc.subjectPhosphatidate phosphatase
dc.subjectEscherichia coli
dc.subjectOil production
dc.subjectTriacylglycerol
dc.titleEngineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production
dc.typearticulo
dc.type.collectionarticulo
dc.type.versionpublishedVersion

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