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Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

ABSTRACT: UNLABELLED: BACKGROUND:Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microbial lipids production. RESULTS:We cloned and expressed a cytosolic Acinetobacter baylyi thioesterase ('AcTesA) in E. coli by deleting its leader sequence. Protein sequence alignment, structure modeling and site-directed mutagenesis demonstrated that Ser10, Gly48, Asn77, Asp158 and His161 residues composed the active centre of 'AcTesA. The engineered strain that overexpressed 'AcTesA achieved a FFAs titer of up to 501.2 mg/L in shake flask, in contrast to only 20.5 mg/L obtained in wild-type E. coli, demonstrating that the expression of 'AcTesA indeed boosted the synthesis of FFAs. The 'AcTesA exhibited a substrate preference towards the C8-C16 acyl groups, with C14:0, C16:1, C12:0 and C8:0 FFAs being the top four components. Optimization of expression level of 'AcTesA made the FFAs production increase to 551.3 mg/L. The FFAs production further increased to 716.1 mg/L by optimization of the culture medium. Fed-batch fermentation was also carried out to evaluate the FFAs production in a scaleable process. Finally, 3.6 g/L FFAs were accumulated within 48 h, and a maximal FFAs yield of 6.1% was achieved in 12-16 h post induction. CONCLUSIONS:For the first time, an A. baylyi thioesterase was cloned and solubly expressed in the cytosol of E. coli. This leaderless thioesterase ('AcTesA) was found to be capable of enhancing the FFAs production of E. coli. Without detailed optimization of the strain and fermentation, the finally achieved 3.6 g/L FFAs is encouraging. In addition, 'AcTesA exhibited different substrate specificity from other thioesterases previously reported, and can be used to supply the fatty acid-based biofuels with high quality of FFAs. Altogether, this study provides a promising thioesterase for FFAs production, and is of great importance in enriching the library of useful thioesterases.

SUBMITTER: Zheng Y

PROVIDER: S-EPMC3524773 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

Zheng Yanning Y Li Lingling L Liu Qiang Q Qin Wen W Yang Jianming J Cao Yujin Y Jiang Xinglin X Zhao Guang G Xian Mo M

Biotechnology for biofuels 20121011 1

<h4>Unlabelled</h4><h4>Background</h4>Thioesterases remove the fatty acyl moiety from the fatty acyl-acyl carrier proteins (ACPs), releasing them as free fatty acids (FFAs), which can be further used to produce a variety of fatty acid-based biofuels, such as biodiesel, fatty alcohols and alkanes. Thioesterases play a key role in the regulation of the fatty acid synthesis in Escherichia coli. Therefore, exploring more promising thioesterases will contribute to the development of industrial microb ...[more]

PMID: 23057831

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Project description:The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications.IMPORTANCEAcinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits.

Project description:Comparative analysis of changes in gene and protein expression and fatty acid profiles between Escherichia coli K-12 MG1655 ΔfadD ΔaraBAD expressing an acyl-acyl carrier protein thioesterase from Umbellularia californica (BTE) or a non-functional mutant thioesterase (BTE-H204A) to determine the functional basis for losses in cell viability, membrane integrity, or other stresses and metabolic perturbations that may be present. New hypotheses obtained from the study will assist in metabolic engineering efforts of improved strains exhibiting higher fatty acid yields and productivities. Cultures of fatty acid overproducing (BTE-expressing) and negative control (non-functional BTE-H204A-expressing) strains of Escherichia coli K-12 MG1655 delta-fadD delta-araBAD (deficient in beta-oxidation and L-arabinose catabolism) were sampled under two different sets of media/induction/antibiotic conditions. These conditions were shake flasks at 37C, 250 rpm shaking, in EZ rich defined medium supplemented with 0.2% glucose and 0.01 mM biotin (EZglu), and in fermentors at 37C with controlled air sparging, agitation, and pH in EZ rich defined medium supplemented with 0.4% glycerol and 0.01 mM biotin (EZgly). Two strains were analyzed in the EZglu experiment, the background strain harboring either pTrc99A-BTE (fatty acid overproducing) or pTrc99A-BTE-H204A (control phenotype). Three strains were analyzed in the EZgly experiment, with the background strain haboring either pBAD35-BTE and pBAD33 (fatty acid overproducing), pBAD35-BTE and pBAD33-ACC (fatty acid overproducing, ACC are the 4 subunits of E. coli K-12 acetyl-CoA carboxylase expressed as an artificial operon accDABC in plasmid pBAD33), and pBAD35-BTE-H204A and pBAD33 (control phenotype). RNA was extracted from harvested cell pellets from biological triplicates (EZglu) or biological duplicates (EZgly) of each strain at three different sampling times as defined in each sample description. Due to a hybridization or scanning problem, biological duplicates rather than triplicates were analyzed at the mid-stationary phase sampling point in the EZglu experiment for the control strain harboring pTrc99A-BTE-H204A. Multiple technical replicates at either the hybridization or sample level were analyzed from the biological duplicates of the fermentor experiment. The form of technical replicate (sample or hybridization) is specified in each EZgly sample description.

Dataset Information

Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

Publications

Boosting the free fatty acid synthesis of Escherichia coli by expression of a cytosolic Acinetobacter baylyi thioesterase.

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