Purification, Characterization, and Potential Bacterial Wax Production Role of an NADPH-Dependent Fatty Aldehyde Reductase from Marinobacter aquaeolei VT8

Abstract

Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/min/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP ؉ . The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed.

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

“…Besides our earlier report expressing a Mus musculus FAR in S. cerevisiae (Runguphan and Keasling, 2013), other labs have reported on heterologously expressing a FAR from Tyto alba (Feng et al, 2015) and two from Marinobacter hydrocarbonasticus (Liu et al, 2013;Wahlen et al, 2009;Willis et al, 2011). To determine the variant most effective for fatty alcohol production in S. cerevisiae, we chromosomally integrated the four FAR coding sequences (MmFAR1, TaFAR1, MaFAR2, and MaFAR7, codon-optimized sequences in Suppl.…”

Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

“…While the fatty acyl-CoA utilized by the WS/ DGAT is proposed to come directly from the fatty acyl-CoA pool, the fatty alcohol is believed to be produced through the action of several reductase enzymes acting on activated fatty acids or fatty aldehydes. M. aquaeolei VT8 contains at least two enzymes that have been found to produce fatty alcohols from several different substrates in vitro, including fatty aldehydes, fatty acyl-CoAs, and fatty acyl-acyl carrier proteins (ACPs) (4)(5)(6)(7). Additionally, both of the isolated enzymes from M. aquaeolei VT8 have significantly higher activities than those reported for other enzymes when tested in in vitro assays versus the enzyme isolated from Acinetobacter (4,(6)(7)(8).…”

Purification and Characterization of an NAD + -Dependent XylB-Like Aryl Alcohol Dehydrogenase Identified in Acinetobacter baylyi ADP1

“…Coenzyme A-activated fatty acids are therein reduced by the NADPH-dependent acyl-CoA reductase Acr1 (annotation number ACIAD3383) (23) to fatty aldehydes, which are proposed to be further reduced to the corresponding fatty alcohol. However, the enzyme responsible for the latter step in A. baylyi ADP1 has not been identified yet, although Tani et al (31) suggested the NADPH-dependent alcohol dehydrogenase AlrA (ACIAD3612) to be responsible for this reaction in A. baylyi, and aldehydereducing enzymes have been recently identified in Marinobacter aquaeolei VT8 (33). Finally, the fatty alcohol is condensed with the fatty acyl moiety of the CoA derivative by the promiscuous wax ester synthase/diacylglycerol:acetyltransferase (WS/ DGAT) (ACIAD0832) to form a wax ester.…”