Abstract
BackgroundMicrobial 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.ResultsThe 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0172-0) contains supplementary material, which is available to authorized users.
Highlights
Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries
Arabolaza et al showed that the expression of the diacylglycerol acyltransferase (DGAT) Sco0958 in an E. coli strain containing a deletion in the dgkA gene is sufficient to induce TAG accumulation [15], suggesting that DAG is the limiting metabolite for TAG biosynthesis in E. coli
To test if DAG was still a limiting factor in a dgkA background, we evaluated the impact of Lppβ in TAG biosynthesis in a BL21 ΔdgkA strain - named strain MPS11 - in the presence of Sco0958
Summary
Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Microorganisms that naturally synthesize TAG (like species of Rhodococcus, Mycobacterium, and Streptomyces genera) can reach high levels of this neutral lipid under specific growth conditions, as is the case of R. opaccus grown on gluconate medium which is capable of accumulating TAG accounting for up to 76% of the cell dry weight (CDW) [1,7]. These microorganisms generally exhibit a rather slow growth rate and may require substantial and challenging genetic modifications for higher TAG productivity and/or for substrate utilization [8]. Deletion of dgkA impairs the recycling of the DAG generated during the synthesis of membrane-derived oligosaccharides, leading to the accumulation of the DAG moiety [13,14]
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