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1
المؤلفون: Don R. Ranatunga, Wesley D. Marner, Rebecca M. Lennen, William Bothfeld, Brian F. Pfleger, J. Tyler Youngquist
المصدر: Biotechnology and Bioengineering. 109:1518-1527
مصطلحات موضوعية: chemistry.chemical_classification, Strain (chemistry), Fatty acid, Bioengineering, Chemostat, Fatty Acids, Nonesterified, Models, Theoretical, Biology, medicine.disease_cause, Applied Microbiology and Biotechnology, Recombinant Proteins, Dilution, Glucose, Palmitoyl-CoA Hydrolase, chemistry, Thioesterase, Biochemistry, Biofuels, Yield (chemistry), Umbellularia, Escherichia coli, medicine, Growth rate, Biotechnology
الوصف: The microbial production of free fatty acids (FFAs) and reduced derivatives is an attractive process for the renewable production of diesel fuels. Toward this goal, a plasmid-free strain of Escherichia coli was engineered to produce FFAs by integrating three copies of a thioesterase gene from Umbellularia californica (BTE) under the control of an inducible promoter onto the chromosome. In batch culture, the resulting strain produced identical titers to a previously reported strain that expressed the thioesterase from a plasmid. The growth rate, glucose consumption rate, and FFA production rate of this strain were studied in continuous cultivation under carbon limitation. The highest yield of FFA on glucose was observed at a dilution rate of 0.05 h(-1) with the highest specific productivity observed at a dilution rate of 0.2 h(-1). The observed yields under the lowest dilution rate were 15% higher than that observed in batch cultures. An increase in both productivity and yield (≈ 40%) was observed when the composition of the nutrients was altered to shift the culture toward non-carbon limitation. A deterministic model of the production strain has been proposed and indicates that maintenance requirements for this strain are significantly higher than wild-type E. coli.
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2
المؤلفون: Brian F. Pfleger, Matthew F. Copeland, Martin H. Schumacher, Mark C. Politz, J. Tyler Youngquist, Thomas C. Raines, Joshua P. Rose
المصدر: Metabolic engineering. 20
مصطلحات موضوعية: Commodity chemicals, Fatty alcohol, Bioengineering, Biology, medicine.disease_cause, Applied Microbiology and Biotechnology, Article, Metabolic engineering, chemistry.chemical_compound, Thioesterase, Bacterial Proteins, medicine, Escherichia coli, Aldehyde Reductase, Pseudomonas putida, Recombinant Proteins, Glucose, chemistry, Biochemistry, Metabolic Engineering, Dodecanol, Fermentation, Fatty Alcohols, Biotechnology
الوصف: Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol / g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.
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3
المصدر: Metabolic engineering. 14(6)
مصطلحات موضوعية: Glucose, Multienzyme Complexes, Polyhydroxyalkanoates, Pseudomonas aeruginosa, Escherichia coli, lipids (amino acids, peptides, and proteins), Cloning, Molecular, Protein Engineering, Recombinant Proteins, Article
الوصف: Demand for sustainable materials motivates the development of microorganisms capable of synthesizing products from renewable substrates. A challenge to commercial production of polyhydroxyalkanoates (PHA), microbially derived polyesters, is engineering metabolic pathways to produce a polymer with the desired monomer composition from an unrelated and renewable source. Here, we demonstrate a metabolic pathway for converting glucose into medium-chain-length (mcl)-PHA composed primarily of 3-hydroxydodecanoate monomers. This pathway combines fatty acid biosynthesis, an acyl-ACP thioesterase to generate desired C12 and C14 fatty acids, β-oxidation for conversion of fatty acids to (R)-3-hydroxyacyl-CoAs, and a PHA polymerase. A key finding is that Escherichia coli expresses multiple copies of enzymes involved in β-oxidation under aerobic conditions. To produce polyhydroxydodecanoate, an acyl-ACP thioesterase (BTE), an enoyl-CoA hydratase (phaJ3), and mcl-PHA polymerase (phaC2) were overexpressed in E. coli ΔfadRABIJ. Yields were improved through expression of an acyl-CoA synthetase resulting in production over 15% CDW – the highest reported production of mcl-PHA of a defined composition from an unrelated carbon source.
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