INVESTIGADORES
GRAMAJO Hugo Cesar
artículos
Título:
Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production
Autor/es:
SANTIAGO COMBA, MARTÍN SABATINI, SIMÓN MENENDEZ-BRAVO, ANA ARABOLAZA, AND HUGO GRAMAJO
Revista:
BIOTECHNOLOGY FOR BIOFUELS
Editorial:
BIOMED CENTRAL LTD
Referencias:
Lugar: Londres; Año: 2014
ISSN:
1754-6834
Resumen:
Abstract
Background: 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.