IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
artículos
Título:
Engineering a Streptomyces coelicolor biosynthesis pathway into Escherichia coli for high yield triglyceride production
Autor/es:
COMBA, SANTIAGO; SABATINI, MARTIN; MENENDEZ-BRAVO, SIMÓN; ARABOLAZA, ANA; GRAMAJO, HUGO
Revista:
BIOTECHNOLOGY FOR BIOFUELS
Editorial:
BIOMED CENTRAL LTD
Referencias:
Lugar: Londres; Año: 2014 vol. 7
ISSN:
1754-6834
Resumen:
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.