Abstract
It has been previously reported that photosynthetic production of extracellular free fatty acids (FFAs) in cyanobacteria was realized by thioesterases (TesA) mediated hydrolysis of fatty acyl-ACP in cytosol and excretion of the FFA outside of the cell. However, two major issues related to the genetically modified strains need to be addressed before the scale-up commercial application becomes possible: namely, the toxicity of FFAs, and the diversity of carbon lengths of fatty acids that could mimic the fossil fuel. To address those issues, we hypothesized that generating FFAs near membrane could facilitate rapid excretion of the FFA outside of the cell and thus decrease toxicity caused by intracellular FFAs in the cytosolic expression of thioesterase. To realize this, we localized a leaderless thioesterase (AcTesA) from Acinetobacter baylyi on the cytosolic side of the inner membrane of Synechocystis sp. PCC6803 using a membrane scaffolding system. The engineered strain with AcTesA on its membrane (mAcT) produced extracellular FFAs up to 171.9 ± 13.22 mg⋅L-1 compared with 40.24 ± 10.94 and 1.904 ± 0.158 mg⋅L-1 in the cytosol-expressed AcTesA (AcT) and wild-type (WT) strains, respectively. Moreover, the mAcT strain generated around 1.5 and 1.9 times less reactive oxygen species than AcT and WT, respectively. Approximately 78% of total FFAs were secreted with an average rate of 1 mg⋅L-1⋅h-1, which was higher than 0.44 mg⋅L-1⋅h-1 reported previously. In the case of mAcT strain, 60% of total secreted FFAs was monounsaturated (C18:1) which is the preferable biodiesel component. Therefore, the engineered mAcT strain shows enhanced FFAs production with less toxicity which is highly desirable for biodiesel production.
Highlights
Given their rapid growth, simple nutrient requirements, ability to incorporate environmental CO2 and solar energy, and the recent progress in understanding physiology at the molecular level, cyanobacteria offer a promising host system for fatty acids based biodiesel production as an alternative to fossil and algal fuels (Lardon et al, 2009; Hannon et al, 2010; Liu et al, 2011b; Jin et al, 2014)
Acinetobacter baylyi was reported to have four different thioesterases, namely, TesA, B, C and D
This AcTesA from Acinetobacter baylyi composed of 212 amino acids (AA) in length, which includes a 30 AA-long N-terminus leader sequence that helps the native protein to localize in the periplasmic space of cells (Zheng et al, 2012)
Summary
Simple nutrient requirements, ability to incorporate environmental CO2 and solar energy, and the recent progress in understanding physiology at the molecular level, cyanobacteria offer a promising host system for fatty acids based biodiesel production as an alternative to fossil and algal fuels (Lardon et al, 2009; Hannon et al, 2010; Liu et al, 2011b; Jin et al, 2014). Biodiesel must adopt the quality of fossil fuel before it can be used in currently available diesel engines. This quality mostly depends on the degree of saturation/unsaturation in the carbon length of fatty acid ester in biodiesel. An ideal biodiesel would be a mixture of both saturated and monounsaturated fatty acid esters with cetane value of more than 40 (Cao et al, 2014). The heterologous expression of thioesterase hydrolyses acyl-ACP to generate free fatty acids (FFAs) and secrete them out of cells in cyanobacteria. Despite the success in expressing different thioesterases and the production of FFAs in cyanobacteria, the effort is still needed to produce unsaturated FFAs in an engineered host with minimum toxic effect on it to achieve practical application
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