Acinetobacter calcoaceticus BD413 accumulates wax esters and triacylglycerol under conditions of mineral nutrient limitation. Nitrosoguanidine-induced mutants of strain BD413 were isolated that failed to accumulate wax esters under nitrogen-limited growth conditions. One of the mutants, Wow15 (without wax), accumulated wax when grown in the presence of cis-11-hexadecenal and hexadecanol but not hexadecane or hexadecanoic acid. This suggested that the mutation may have inactivated a gene encoding either an acyl-acyl carrier protein or acyl-coenzyme A (CoA) reductase. The Wow15 mutant was complemented with a cosmid genomic library prepared from wild-type A. calcoaceticus BD413. The complementary region was localized to a single gene (acr1) encoding a protein of 32,468 Da that is 44% identical over a region of 264 amino acids to a product of unknown function encoded by an open reading frame associated with mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. Extracts of Escherichia coli cells expressing the acr1 gene catalyzed the reduction of acyl-CoA to the corresponding fatty aldehyde, indicating that the gene encodes a novel fatty acyl-CoA reductase.Wax esters are utilized in diverse biological roles. Prominent uses include coating the surfaces of plant leaves as epicuticular waxes to provide desiccation tolerance and protection against ultraviolet light and serving as carbon storage inclusions in some bacterial species such as Acinetobacter calcoaceticus. Wax esters also have industrial applications, serving as high-temperature lubricants, surface coatings, and emulsifying agents.The chemical structures of wax esters produced by A. calcoaceticus are similar to those found in other organisms such as jojoba (Simmondsia chinensis L.), a plant species native to the southwest region of the United States that is used for commercial wax ester production, and the sperm whale, the commercial source of wax esters prior to the worldwide ban on whaling (7). The proposed pathway for wax ester biosynthesis in A. calcoaceticus is illustrated in Fig. 1. In this pathway, three enzymes are directly involved in the conversion of acyl-acyl carrier protein (ACP), or acyl-coenzyme A (CoA), to wax esters. In the first step, acyl-CoA (or acyl-ACP) is reduced by an acyl-CoA (or acyl-ACP) reductase to its corresponding fatty aldehyde. This fatty aldehyde is then further reduced to its corresponding fatty alcohol by a fatty aldehyde reductase. Finally, an acyl-CoA (acyl-ACP) fatty alcohol transferase condenses the fatty alcohol with either acyl-ACP or acyl-CoA to give the final wax ester product. The first two steps of this pathway are indirectly supported by biochemical evidence gathered from studies of the metabolism of alkanes by Acinetobacter species and from studying wax ester biosynthesis in other microorganisms (10).Several previous studies have reported that fatty alcohol and fatty aldehyde dehydrogenases exist in various Acinetobacter species (6,11,24,25). These observations have been the result of investigations into th...

Poly(hydroxyalkanoates) are natural polymers with thermoplastic properties. One polymer of this class with commercial applicability, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) can be produced by bacterial fermentation, but the process is not economically competitive with polymer production from petrochemicals. Poly(hydroxyalkanoate) production in green plants promises much lower costs, but producing copolymer with the appropriate monomer composition is problematic. In this study, we have engineered Arabidopsis and Brassica to produce PHBV in leaves and seeds, respectively, by redirecting the metabolic flow of intermediates from fatty acid and amino acid biosynthesis. We present a pathway for the biosynthesis of PHBV in plant plastids, and also report copolymer production, metabolic intermediate analyses, and pathway dynamics.

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Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760-12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a beta-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(beta-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation.