Abstract
BackgroundFatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds.ResultsFatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters.ConclusionsThe fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.
Highlights
Fatty aldehydes are industrially relevant compounds, which represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters
A preliminary in vivo characterization of Ramo in E. coli indicated fatty aldehyde production associated with its expression (Additional file 1)
In order to determine whether the overexpression of Acr1 or Ramo has an effect on the carbon chain length, we studied the composition of the wax esters (WE) with NMR and gas chromatography
Summary
Fatty aldehydes are industrially relevant compounds, which represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. For the production of the long carbon chains required in oleochemicals, the fatty acid biosynthetic pathway is one of the few existing metabolic pathways [2] Fatty acids and their activated forms (fatty acyl-CoAs and -ACPs) are precursors for a range of industrially relevant products, including alkanes, fatty. The key enzymes in aldehyde synthesis are fatty acyl-CoA (or -ACP) reductases (FAR). Various such reductases have been studied, including Aar from Synechococcus elongatus PCC 7942 [6], Aar-homologs from other cyanobacteria [7], and Acr from Acinetobacter baylyi ADP1 [8]. The cultivation time (48 h) is long enough to ensure that all the cultures have reached stationary phase at the time of WE analysis
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