In this study, we evaluated the effects of several metabolic
engineering
strategies in a systematic and combinatorial manner to enhance the
free fatty acid (FFA) production in Escherichia coli. The strategies included (i) overexpression of mutant thioesterase
I (‘TesAR64C) to efficiently release the FFAs from
fatty acyl-ACP; (ii) coexpression of global regulatory protein FadR;
(iii) heterologous expression of methylmalonyl-CoA carboxyltransferase
and phosphoenolpyruvate carboxylase to synthesize fatty acid precursor
molecule malonyl-CoA; and (iv) disruption of genes associated with
membrane proteins (GusC, MdlA, and EnvR) to improve the cellular state
and export the FFAs outside the cell. The synergistic effects of these
genetic modifications in strain SBF50 yielded 7.2 ± 0.11 g/L
FFAs at the shake flask level. In fed-batch cultivation under nitrogen-limiting
conditions, strain SBF50 produced 33.6 ± 0.02 g/L FFAs with a
productivity of 0.7 g/L/h from glucose, which is the maximum titer
reported in E. coli to date. Combinatorial
metabolic engineering approaches can prove to be highly useful for
the large-scale production of FA-derived chemicals and fuels.
In this study, whole‐cell biotransformation was conducted to produce nonanedioic acid from nonanoic acid by expressing the alkane hydroxylating system (AlkBGT) from Pseudomonas putida GPo1 in Escherichia coli. Following adaptive laboratory evolution, an efficient E. coli mutant strain, designated as MRE, was successfully obtained, demonstrating the fastest growth (27‐fold higher) on nonanoic acid as the sole carbon source compared to the wild‐type strain. Additionally, the MRE strain was engineered to block nonanoic acid degradation by deleting fadE. The resulting strain exhibited a 12.8‐fold increase in nonanedioic acid production compared to the wild‐type strain. Six mutations in acrR, Pcrp, dppA, PfadD, e14, and yeaR were identified in the mutant MRE strain, which was characterized using genomic modifications and RNA‐sequencing. The acquired mutations were found to be beneficial for rapid growth and nonanedioic acid production.
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