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Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

ABSTRACT: Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42 degrees C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.

SUBMITTER: Rock CO

PROVIDER: S-EPMC178354 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

Rock C O CO Tsay J T JT Heath R R Jackowski S S

Journal of bacteriology 19960901 18

Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. Howev ...[more]

PMID: 8808925

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Project description:Hydroxy fatty acids (HFAs) are valuable chemicals for a broad variety of applications. However, commercial production of HFAs has not been established so far due to the lack of low cost routes for their synthesis. Although the microbial transformation pathway of HFAs was extensively studied decades ago, these attempts mainly focused on converting fatty acids or vegetable oils to their hydroxyl counterparts. The use of a wider range of feedstocks to produce HFAs would reduce the dependence on oil crops and be expected to cut down the manufacturing cost.In this study, the industrially important microorganism Escherichia coli was engineered to produce HFAs directly from glucose. Through the coexpression of the acetyl-CoA carboxylase (ACCase) and the leadless acyl-CoA thioesterase ('TesA), and knockout of the endogenous acyl-CoA synthetase (FadD), an engineered E. coli strain was constructed to efficiently synthesize free fatty acids (FFAs). Under shake-flask conditions, 244.8 mg/L of FFAs were obtained by a 12 h induced culture. Then the fatty acid hydroxylase (CYP102A1) from Bacillus megaterium was introduced into this strain and high-level production of HFAs was achieved. The finally engineered strain BL21?fadD/pE-A1'tesA&pA-acc accumulated up to 58.7 mg/L of HFAs in the culture broth. About 24 % of the FFAs generated by the thioesterase were converted to HFAs. Fatty acid composition analysis showed that the HFAs mainly consisted of 9-hydroxydecanoic acid (9-OH-C10), 11-hydroxydodecanoic acid (11-OH-C12), 10-hydroxyhexadecanoic acid (10-OH-C16) and 12-hydroxyoctadecanoic acid (12-OH-C18). Fed-batch fermentation of this strain further increased the final titer of HFAs to 548 mg/L.A robust HFA-producing strain was successfully constructed using glucose as the feedstock, which demonstrated a novel strategy for bioproduction of HFAs. The results of this work suggest that metabolically engineered E. coli has the potential to be a microbial cell factory for large-scale production of HFAs.

Dataset Information

Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

Publications

Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

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