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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